*3.5.2 Determination of component participating in larval growth*

In order to ascertain the RJ component responsible for larval growth [27], compositions of proteins and 10-HDA (considered to be a tool for functional ingredients for activity) were compared during the cultivation.

As shown in **Figure 15**, the contents of total proteins decreased during the cultivation, whereas there was a slight change in the percentage of 10-HDA. These results indicate the possibility that proteins are consumed by the larvae as they grow.

In order to further confirm the result, elution profiles of soluble RJ proteins by size-exclusion HPLC on Superose 12 column were compared during cultivation. As

shown in **Figure 16**, crude soluble RJ proteins were separated as five peaks,

In order to further confirm the participation of Peak 2 protein (MRJP-1 multimer) in larval growth, the body weight of larvae cultured in RJ was compared

*Elution profile of soluble protein in RJ. A typical elution pattern of soluble RJ proteins. Peak 2 represents MRJP-*

oligomeric form, MRJP-1 multimer (**Figure 17**).

*DOI: http://dx.doi.org/10.5772/intechopen.89647*

**Figure 15.**

**Figure 16.**

*1 multimer.*

**143**

with RJ samples that had different content of Peak 2 proteins.

although Peak 1 and Peak 2 overlapped. Each peak was estimated at about 640 kDa (Peak 1), 360 kDa (Peak 2), 100 kDa (Peak 3), 72 kDa (Peak 4), and 4.5 kDa (Peak 5) in their molecular size, respectively. These five peaks were universally detected in all RJ samples examined. The peak 2 protein was considered to be

*Changes to major components, total protein and 10-HDA in RJ during the cultivation of larval growth in vitro. The total protein and 10-HDA in RJ were analyzed in a chamber filled with 750 ml of fresh RJ and grafted second-instar worker bee larvae. Total protein and 10-HDA were analyzed for the indicated periods.*

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping…*

#### **Figure 14.**

*Representative data of larval growth cultured in RJ in vitro. Second-instar worker bee larvae were grafted in a 12-well flat-bottomed plastic plate filled with 750 mg fresh RJ. The plate was then set in an incubation chamber and cultured at 31°C for the appropriate period of time.*

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping… DOI: http://dx.doi.org/10.5772/intechopen.89647*

#### **Figure 15.**

The growth of larvae at Day 0, Day 3 and Day 6 is also represented in **Figure 14**.

*The growth of larvae in RJ in vitro. Second-instar worker bee larvae were grafted in a 12-well flat-bottomed plastic plate filled with 750 mg fresh RJ. The plate was then set in an incubation chamber and cultured at 31°C*

In order to ascertain the RJ component responsible for larval growth [27], compositions of proteins and 10-HDA (considered to be a tool for functional ingredients

In order to further confirm the result, elution profiles of soluble RJ proteins by size-exclusion HPLC on Superose 12 column were compared during cultivation. As

*Representative data of larval growth cultured in RJ in vitro. Second-instar worker bee larvae were grafted in a 12-well flat-bottomed plastic plate filled with 750 mg fresh RJ. The plate was then set in an incubation*

As shown in **Figure 15**, the contents of total proteins decreased during the cultivation, whereas there was a slight change in the percentage of 10-HDA. These results indicate the possibility that proteins are consumed by the larvae as they

It was also confirmed that the larvae grew rapidly in the RJ in vitro [26].

*for appropriate period. Dotted line: RJ harvested at 48 h. Solid line: RJ harvested on 72 h.*

*3.5.2 Determination of component participating in larval growth*

for activity) were compared during the cultivation.

*Modern Beekeeping - Bases for Sustainable Production*

*chamber and cultured at 31°C for the appropriate period of time.*

grow.

**Figure 14.**

**142**

**Figure 13.**

*Changes to major components, total protein and 10-HDA in RJ during the cultivation of larval growth in vitro. The total protein and 10-HDA in RJ were analyzed in a chamber filled with 750 ml of fresh RJ and grafted second-instar worker bee larvae. Total protein and 10-HDA were analyzed for the indicated periods.*

shown in **Figure 16**, crude soluble RJ proteins were separated as five peaks, although Peak 1 and Peak 2 overlapped. Each peak was estimated at about 640 kDa (Peak 1), 360 kDa (Peak 2), 100 kDa (Peak 3), 72 kDa (Peak 4), and 4.5 kDa (Peak 5) in their molecular size, respectively. These five peaks were universally detected in all RJ samples examined. The peak 2 protein was considered to be oligomeric form, MRJP-1 multimer (**Figure 17**).

In order to further confirm the participation of Peak 2 protein (MRJP-1 multimer) in larval growth, the body weight of larvae cultured in RJ was compared with RJ samples that had different content of Peak 2 proteins.

**Figure 16.** *Elution profile of soluble protein in RJ. A typical elution pattern of soluble RJ proteins. Peak 2 represents MRJP-1 multimer.*

**Figure 17.**

*A case observed where peak 2 disappeared in 72-h cultivation. An unusual case where peak 2 disappeared at 72-h cultivation.*

Although crude soluble RJ proteins are distinguished in five peaks, the level of Peak 2 (MRJP-1 multimer) varied greatly in some RJ samples, as seen in **Figure 18A**.

The growth of larvae between RJ samples with high and low Peak 2 level was compared and the results are expressed in **Figure 18B**. The growth of larvae was greater in the Peak 2-rich RJ samples than in samples with poor Peak 2 poor. These results further confirmed that the Peak 2 proteins are a key substance in the growth and development of honey bee queens.

#### *3.5.3 The content of peak 2 protein (MRJP-1 Multimer) in queen cell and its stability*

During the study of Peak 2 protein, I confirmed that this protein is a MRJP-1 multimer [26].

Next, the transitional change of MRJP-1 multimer content was monitored in artificial queen cells in order to investigate the relation between MRJP-1 multimer contents and the duration from setting of the queen cells to the harvesting of RJ. As seen in **Figure 19**, MRJP-1 multimer contents decreased gradually over time after setting the queen cells. In the previous experiment in which larvae were cultured in a plastic plate filled with RJ, the author postulated that MRJP-1 multimer was consumed and decreased as larvae grew (**Figure 17**).

Although a remarkable decrease of MRJP-1 multimer as in the previous experiment was not observed, the MRJP-1 multimer content was found to decrease according to the larval growth, that is, time elapsed after queen cell setting, due to the addition of fresh RJ by worker bees.

of worker bees should be guaranteed by rotating the beehives employed for RJ production, as well as by limiting the number of artificial queen cells per colony.

*Larval growth in MRJP-1 Multimer-rich and MRJP-1 Multimer-poor samples of RJ. Larval growth and body weight of larvae cultured in RJ were compared among RJ samples which were different in content of peak 2 proteins. (A) Growth of larvae in MRJP-1 multimer rich RJ (*●*) and MRJP-1 poor RJ (*○*), which correspond*

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping…*

*DOI: http://dx.doi.org/10.5772/intechopen.89647*

Since the essential function of RJ is to produce larval growth and ensure differential development of a queen bee, development of larvae in RJ was investigated and components participating in the function were identified. The proteins, especially Peak 2 protein (MRJP-1 multimer), diminished as the larva grew. The growth of larvae was better in the Peak 2-rich RJ samples than in the Peak 2-poor RJ, indicating that the Peak 2 protein (MRJP-1 multimer) might be the most important substance of RJ. The results suggest that the quality of RJ may change by different period for harvesting and size/age of larvae transferred. In addition, the results suggest the possibility that the content of Peak 2 protein (MRJP-1 multimer) differs depending on the size of larvae transferred and the duration from larval transfer to harvest of RJ, and that the quality of RJ is not necessarily uniform among samples

The results also support my proposal for production of high-quality RJ.

*3.5.4 Discussion and summary*

*to the right and left figures in (B), respectively.*

**Figure 18.**

**145**

produced under varying conditions.

The MRJP-1 multimer content was shown to decrease by about 30% decrease 74 h after larval graft to queen cells. The results indicate that the quality of RJ may decrease together with the passing of time until harvest even under conditions in which fresh RJ is successively appended to queen cells by worker bees. The results also support the view that RJ harvested 48 h after queen cell setting provides higher quality than RJ that is harvested after 72 h. Moreover, MRJP-1 multimer contents among the RJ samples were compared with and without rotation of RJ production by worker bees, and the results are shown in **Figure 11**. The MRJP-1 multimer content was significantly higher in 48 h-harvested RJ by worker bees rested 2 days before the RJ production than in 72 h-harvested RJ successively produced by worker bees without resting rotation (p < 0.05). The amount of 10-HDA in these RJ samples was also compared in **Figure 11** and it was found that 10-HDA content was significantly higher in 48-h harvested RJ than in 72 h-harvested RJ (p < 0.05). I have emphasized in the author's proposal on natural beekeeping that the health

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping… DOI: http://dx.doi.org/10.5772/intechopen.89647*

#### **Figure 18.**

Although crude soluble RJ proteins are distinguished in five peaks, the level of Peak 2 (MRJP-1 multimer) varied greatly in some RJ samples, as seen in **Figure 18A**. The growth of larvae between RJ samples with high and low Peak 2 level was compared and the results are expressed in **Figure 18B**. The growth of larvae was greater in the Peak 2-rich RJ samples than in samples with poor Peak 2 poor. These results further confirmed that the Peak 2 proteins are a key substance in the growth

*A case observed where peak 2 disappeared in 72-h cultivation. An unusual case where peak 2 disappeared at*

*3.5.3 The content of peak 2 protein (MRJP-1 Multimer) in queen cell and its stability*

During the study of Peak 2 protein, I confirmed that this protein is a MRJP-1

Next, the transitional change of MRJP-1 multimer content was monitored in artificial queen cells in order to investigate the relation between MRJP-1 multimer contents and the duration from setting of the queen cells to the harvesting of RJ. As seen in **Figure 19**, MRJP-1 multimer contents decreased gradually over time after setting the queen cells. In the previous experiment in which larvae were cultured in a plastic plate filled with RJ, the author postulated that MRJP-1 multimer was

Although a remarkable decrease of MRJP-1 multimer as in the previous experi-

The MRJP-1 multimer content was shown to decrease by about 30% decrease 74 h after larval graft to queen cells. The results indicate that the quality of RJ may decrease together with the passing of time until harvest even under conditions in which fresh RJ is successively appended to queen cells by worker bees. The results also support the view that RJ harvested 48 h after queen cell setting provides higher quality than RJ that is harvested after 72 h. Moreover, MRJP-1 multimer contents among the RJ samples were compared with and without rotation of RJ production by worker bees, and the results are shown in **Figure 11**. The MRJP-1 multimer content was significantly higher in 48 h-harvested RJ by worker bees rested 2 days before the RJ production than in 72 h-harvested RJ successively produced by worker bees without resting rotation (p < 0.05). The amount of 10-HDA in these RJ samples was also compared in **Figure 11** and it was found that 10-HDA content was significantly higher in 48-h harvested RJ than in 72 h-harvested RJ (p < 0.05). I have emphasized in the author's proposal on natural beekeeping that the health

ment was not observed, the MRJP-1 multimer content was found to decrease according to the larval growth, that is, time elapsed after queen cell setting, due to

and development of honey bee queens.

*Modern Beekeeping - Bases for Sustainable Production*

consumed and decreased as larvae grew (**Figure 17**).

the addition of fresh RJ by worker bees.

multimer [26].

**144**

**Figure 17.**

*72-h cultivation.*

*Larval growth in MRJP-1 Multimer-rich and MRJP-1 Multimer-poor samples of RJ. Larval growth and body weight of larvae cultured in RJ were compared among RJ samples which were different in content of peak 2 proteins. (A) Growth of larvae in MRJP-1 multimer rich RJ (*●*) and MRJP-1 poor RJ (*○*), which correspond to the right and left figures in (B), respectively.*

of worker bees should be guaranteed by rotating the beehives employed for RJ production, as well as by limiting the number of artificial queen cells per colony. The results also support my proposal for production of high-quality RJ.

#### *3.5.4 Discussion and summary*

Since the essential function of RJ is to produce larval growth and ensure differential development of a queen bee, development of larvae in RJ was investigated and components participating in the function were identified. The proteins, especially Peak 2 protein (MRJP-1 multimer), diminished as the larva grew. The growth of larvae was better in the Peak 2-rich RJ samples than in the Peak 2-poor RJ, indicating that the Peak 2 protein (MRJP-1 multimer) might be the most important substance of RJ. The results suggest that the quality of RJ may change by different period for harvesting and size/age of larvae transferred. In addition, the results suggest the possibility that the content of Peak 2 protein (MRJP-1 multimer) differs depending on the size of larvae transferred and the duration from larval transfer to harvest of RJ, and that the quality of RJ is not necessarily uniform among samples produced under varying conditions.

employment, and edification for production of quality-added products and related

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping…*

On the other hand, beekeeping is primarily an agricultural industry, so it is impossible for beekeeping to ignore the aspect of gaining profits from bee products. From this point of view, the author proposed an ideal situation for natural organic beekeeping, based on the idea that this may result in production of high-quality bee products with added value (albeit with less focus on profitability), thereby increasing revenue and guaranteeing the sustainability of beekeeping that has future potential. Thus, the author proposed a novel method, Natural Beekeeping, based on

The functionality and components participating in the function of RJ products

Since the essential function of RJ is to produce larval growth and ensure differential development of a queen bee, development of larvae in RJ was investigated and components participating in the function were identified. The proteins, especially MRJP-1 multimer, diminished as the larva grew. The growth of larvae was better in the MRJP-1 multimer-rich RJ samples than in the MRJP-1 multimer-poor RJ, indicating that the MRJP-1 multimer might be the most important substance of RJ. The quality of RJ may change by different period for harvesting and size/age of larvae transferred. In addition, the content of MRJP-1 multimer differs depending on the size of larvae transferred and the duration from larval transfer to harvest of RJ.

The larval growth of honeybees was largely affected by MRJP-1 multimer content. The content of MRJP-1 multimer varied predominantly among the soluble RJ proteins by each sample produced under various conditions, such as rotation of bee colonies employed for RJ production, harvesting 48 h after setting queen cells, appropriate storage at low temperature, and so on. Naturally well-controlled bee culture should be promoted for sustainable innovation in modern beekeeping to guarantee high quality levels for RJ and other bee products. Also the MRJP-1 multimer content in RJ should be used as a new criterion for functional quality evaluation and as a freshness param-

In conclusion the content of 10HDA and MRJP1 multimer in RJ prepared by Kikuji Yamaguchi Method of Natural Beekeeping (KYAMENABEE) were significantly higher than that prepared by ordinal beekeeping. The biological and pharmacological activities were also superior for RJ prepared by KYAMENABEE than that by ordinal beekeeping. Thus, it might be important to use a novel beekeeping method, KYAMENABEE, in order to produce high quality RJ for sustainable devel-

I am deeply grateful to Prof. Jozef Šimúth and Dr. Katarina Bíliková, Institute of

Forest Ecology, Slovak Academy of Sciences, Zvolen, Department of Molecular Apidology, Bratislava, Slovakia, for their help and valuable suggestions, and Professor Emeritus Akira Fujii, Nihon University, Japan, for his suggestion and help in the preparation of this chapter. I would also like express my sincere thanks to my colleagues, especially to my son Mr. Yoshihisa Yamaguchi, and Dr. Kiyoshi Murata, Ms. Michiyo Takahashi, Mr. Masaki Sakai, Mr. Masakuni Shiomi for their endless help.

produced by this method were studied, and several interesting results were

quality control.

obtained.

the principle of natural beekeeping.

*DOI: http://dx.doi.org/10.5772/intechopen.89647*

eter for RJ, in addition to the use of 10-HDA content.

opment of biopharmaceutical beekeeping.

**Acknowledgements**

**147**

**Figure 19.** *MRJP-1 Multimer content in queen cells after larval graft.*

I demonstrated that the larval growth of honeybees was largely affected by MRJP-1 multimer content. I also proved that the content of MRJP-1 multimer varied predominantly among the soluble RJ proteins by each sample produced under various conditions. The variation of MRJP-1 multimer content is largely affected by the manner of RJ production, such as rotation of bee colonies employed for RJ production, harvesting 48 h after setting queen cells, appropriate storage at low temperature, and so on. The results show that naturally well-controlled bee culture should be promoted for sustainable innovation in modern beekeeping to guarantee high quality levels for RJ and other bee products. Based on these views, the author proposed that the MRJP-1 multimer content in RJ should be used as a new criterion for functional quality evaluation and as a freshness parameter for RJ, in addition to the use of 10-HDA content.

#### **4. Conclusion**

Based on my experience and practice of beekeeping for more than 54 years, the author has postulated many serious problems in recent beekeeping, which should be resolved for sustainable development of industrial beekeeping in the future. The core problems found in modern beekeeping include beekeeping in inappropriate environments, deterioration of colonies due to overloading of production and excessive selective breeding, reduced disease resistance, inappropriate processing, insufficient attention paid to quality control of apicultural products, and so on. Other serious problems also include the deterioration of bee products due to incorrect treatment and inadequate environments for beekeeping, which leads to pollution of bee products due to beekeepers' lack of attention to quality control and the added value of bee products.

In order to resolve these problems, the author proposed that it is essential to recourse to natural organic beekeeping using the natural ability of honey bees, and to make efforts to produce high-quality products by means such as maintenance of appropriate apiary location to prevent pollution through nectar source and water supply, strengthening the activity and ability of honey bees by rotation of beehive

*Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping… DOI: http://dx.doi.org/10.5772/intechopen.89647*

employment, and edification for production of quality-added products and related quality control.

On the other hand, beekeeping is primarily an agricultural industry, so it is impossible for beekeeping to ignore the aspect of gaining profits from bee products. From this point of view, the author proposed an ideal situation for natural organic beekeeping, based on the idea that this may result in production of high-quality bee products with added value (albeit with less focus on profitability), thereby increasing revenue and guaranteeing the sustainability of beekeeping that has future potential. Thus, the author proposed a novel method, Natural Beekeeping, based on the principle of natural beekeeping.

The functionality and components participating in the function of RJ products produced by this method were studied, and several interesting results were obtained.

Since the essential function of RJ is to produce larval growth and ensure differential development of a queen bee, development of larvae in RJ was investigated and components participating in the function were identified. The proteins, especially MRJP-1 multimer, diminished as the larva grew. The growth of larvae was better in the MRJP-1 multimer-rich RJ samples than in the MRJP-1 multimer-poor RJ, indicating that the MRJP-1 multimer might be the most important substance of RJ. The quality of RJ may change by different period for harvesting and size/age of larvae transferred. In addition, the content of MRJP-1 multimer differs depending on the size of larvae transferred and the duration from larval transfer to harvest of RJ.

The larval growth of honeybees was largely affected by MRJP-1 multimer content. The content of MRJP-1 multimer varied predominantly among the soluble RJ proteins by each sample produced under various conditions, such as rotation of bee colonies employed for RJ production, harvesting 48 h after setting queen cells, appropriate storage at low temperature, and so on. Naturally well-controlled bee culture should be promoted for sustainable innovation in modern beekeeping to guarantee high quality levels for RJ and other bee products. Also the MRJP-1 multimer content in RJ should be used as a new criterion for functional quality evaluation and as a freshness parameter for RJ, in addition to the use of 10-HDA content.

In conclusion the content of 10HDA and MRJP1 multimer in RJ prepared by Kikuji Yamaguchi Method of Natural Beekeeping (KYAMENABEE) were significantly higher than that prepared by ordinal beekeeping. The biological and pharmacological activities were also superior for RJ prepared by KYAMENABEE than that by ordinal beekeeping. Thus, it might be important to use a novel beekeeping method, KYAMENABEE, in order to produce high quality RJ for sustainable development of biopharmaceutical beekeeping.
