*3.4.1.3.1 Preparation of sample solution*

Exactly 3 g of RJ kept at 18°C was weighed, thawed at room temperature and diluted with water to make 100 ml. This suspension was stirred well at room temperature for 30 min and then centrifuged for 30 min at 10,000g at 4°C. The supernatant was made to 100 ml using a measuring flask, and 1 ml of the resulting solution was filtered with a PVDF filter 0.22 μm in pore size (Ultrafree-MC, MILLIPORE). The filtrate was used for HPLC analysis.

#### *3.4.1.3.2 HPLC operating conditions*

Column: TSK-gel G3000SW (7.5 mm<sup>Φ</sup> 60 cm: Toso). Mobile phase: 0.1 M Sodium phosphate buffer (pH 7.0) + 0.1 M Na2SO4. Flow rate: 0.6 ml/min. Amount injected: 10 μl. Detector: UV detector (wavelength: 280 nm). Column temperature: Room temperature.

#### *3.4.1.3.3 Calibration line reparation with MRJP-1 multimer standard solutions*

The test was performed with the MRJP-1 multimer standard solutions in line with the "HPLC operating conditions", and the calibration line was prepared from the relation between the peak area and protein concentration (examples of concentration: 0.25, 0.5, 1 and 2 mg/ml).

to queen cells by worker bees. The results also support the opinion that RJ harvested on 48 h after queen cell setting provides higher quality than 72 h-harvested RJ. Moreover, MRJP-1 contents among the RJ samples were compared with or without rotation of RJ production by worker bees and the results are shown in **Figure 11**. The MRJP-1 content was significantly higher in 48-h-harvested RJ (5.72 0.21%) by worker bees rested 2 days before the RJ production than in 72-h-harvested RJ (4.77 0.25%), which was successively produced by worker bees without resting rotation (p < 0.05). The amount of 10-HDA in these RJ samples were also compared in **Figure 11** and was also found to decrease in 72-h-harvested RJ without resting rotation (48-h-harvested RJ, 2.9 0.2%; 72-h-harvested RJ, 2.5 0.1%; p < 0.05). The author has been emphasized in his proposal on natural beekeeping that health of worker bees should be guaranteed by rotation of beehives employed for RJ production, as well as by limiting number of artificial queen cells per colony. The results also support his proposal for production of high-quality RJ.

*Comparison of MRJP-1 Multimer and 10-HDA contents in RJ samples between natural beekeeping (A) and ordinal beekeeping (B) [26]. A: Natural beekeeping (harvested after 48 h, with rotation, n = 9); B: Ordinal*

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

*3.4.2 Reasons why 10-HDA and MRJP-1 multimer are suitable to quality assessment*

10-HDA is the only functional component used for the quality control of RJ (**Table 2**). The author strongly agrees that 10-HDA should be used as the quality control of RJ. Unfortunately, since authentic compound is available and easily added to the product, 10-HDA does not play an important role in the evaluation of RJ. Thus, I proposed one more functional component, MRJP-1 multimer, because this is also a compound only produced by honeybees [28, 30] and it too requires exacting conditions in order to maintain freshness. MRJP-1 is found in a large amount, ca. 60% of soluble protein in RJ, which makes it easy to use as a determining factor. MRJP-1 multimer decomposes easily at high temperature, such as 30°C

The transitional change of MRJP-1 multimer contents stored under different temperatures is shown in **Figure 12**. The MRJP-1 multimer contents decreased depending on temperature and period of storage, with a reduction of about 50% in RJ

and higher, over a period of 2 weeks (**Figure 12**) [26].

*of RJ*

**139**

**Figure 11.**

*beekeeping (harvested after 72 h, without rotation, n = 9).*

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

#### *3.4.1.3.4 Calculation of MRJP-1 multimer content*

Content (%) of MRJP-1 multimer in RJ = concentration (mg/ml) of MRJP-1 multimer obtained from calibration line 1/1000 100 (ml)/3(g) 100.

#### *3.4.1.4 Analytical results of MRJP-1 Multimer and 10-HDA in RJ*

As shown in **Figure 11**, the results of comparing the content of MRJP-1 multimer and 10-HDA in RJ between Natural Beekeeping (harvested after 48 h, with rotation) and ordinal beekeeping (harvested after 72 h, without rotation).

The mean content of MRJP-1 multimer was 5.72 0.21% (n = 9) in the RJ obtained by Natural Beekeeping but 4.77 0.35% (n = 9) in the RJ obtained by ordinal beekeeping, being significantly lower in the latter. Even when compared in unfavorable beekeeping environments, it was confirmed that there were the same level of differences (15–20%) (data not shown).

The mean content of 10-HDA was 2.9 0.2% (n = 9) in the RJ obtained by Natural Beekeeping but 2.5 0.1% (n = 9) in the RJ obtained by ordinal beekeeping, being significantly lower in the latter. Even when compared in unfavorable beekeeping environments, it was confirmed that there were the same level of differences (15–20%) (data not shown).

#### *3.4.1.5 Discussion and summary*

The results indicate that the quality of RJ may decrease according to the time passage until harvest even in the condition in which fresh RJ is successively supplied *Kikuji Yamaguchi Principles of Natural Beekeeping: A Novel Bio-Method of Natural Beekeeping… DOI: http://dx.doi.org/10.5772/intechopen.89647*

#### **Figure 11.**

The RJ was suspended in water. The supernatant obtained after centrifugation of

Exactly 3 g of RJ kept at 18°C was weighed, thawed at room temperature and

diluted with water to make 100 ml. This suspension was stirred well at room temperature for 30 min and then centrifuged for 30 min at 10,000g at 4°C. The supernatant was made to 100 ml using a measuring flask, and 1 ml of the resulting solution was filtered with a PVDF filter 0.22 μm in pore size (Ultrafree-MC,

Mobile phase: 0.1 M Sodium phosphate buffer (pH 7.0) + 0.1 M Na2SO4.

*3.4.1.3.3 Calibration line reparation with MRJP-1 multimer standard solutions*

The test was performed with the MRJP-1 multimer standard solutions in line with the "HPLC operating conditions", and the calibration line was prepared from the relation between the peak area and protein concentration (examples of concen-

Content (%) of MRJP-1 multimer in RJ = concentration (mg/ml) of MRJP-1

As shown in **Figure 11**, the results of comparing the content of MRJP-1 multimer and 10-HDA in RJ between Natural Beekeeping (harvested after 48 h, with rota-

The mean content of MRJP-1 multimer was 5.72 0.21% (n = 9) in the RJ obtained by Natural Beekeeping but 4.77 0.35% (n = 9) in the RJ obtained by ordinal beekeeping, being significantly lower in the latter. Even when compared in unfavorable beekeeping environments, it was confirmed that there were the same

The mean content of 10-HDA was 2.9 0.2% (n = 9) in the RJ obtained by Natural Beekeeping but 2.5 0.1% (n = 9) in the RJ obtained by ordinal beekeeping, being significantly lower in the latter. Even when compared in unfavorable beekeeping environments, it was confirmed that there were the same level of differ-

The results indicate that the quality of RJ may decrease according to the time passage until harvest even in the condition in which fresh RJ is successively supplied

multimer obtained from calibration line 1/1000 100 (ml)/3(g) 100.

tion) and ordinal beekeeping (harvested after 72 h, without rotation).

*3.4.1.4 Analytical results of MRJP-1 Multimer and 10-HDA in RJ*

the suspension was subjected to HPLC analysis.

*Modern Beekeeping - Bases for Sustainable Production*

MILLIPORE). The filtrate was used for HPLC analysis.

Detector: UV detector (wavelength: 280 nm). Column temperature: Room temperature.

Column: TSK-gel G3000SW (7.5 mm<sup>Φ</sup> 60 cm: Toso).

*3.4.1.3.1 Preparation of sample solution*

*3.4.1.3.2 HPLC operating conditions*

Flow rate: 0.6 ml/min. Amount injected: 10 μl.

tration: 0.25, 0.5, 1 and 2 mg/ml).

*3.4.1.3.4 Calculation of MRJP-1 multimer content*

level of differences (15–20%) (data not shown).

ences (15–20%) (data not shown).

*3.4.1.5 Discussion and summary*

**138**

*Comparison of MRJP-1 Multimer and 10-HDA contents in RJ samples between natural beekeeping (A) and ordinal beekeeping (B) [26]. A: Natural beekeeping (harvested after 48 h, with rotation, n = 9); B: Ordinal beekeeping (harvested after 72 h, without rotation, n = 9).*

to queen cells by worker bees. The results also support the opinion that RJ harvested on 48 h after queen cell setting provides higher quality than 72 h-harvested RJ.

Moreover, MRJP-1 contents among the RJ samples were compared with or without rotation of RJ production by worker bees and the results are shown in **Figure 11**. The MRJP-1 content was significantly higher in 48-h-harvested RJ (5.72 0.21%) by worker bees rested 2 days before the RJ production than in 72-h-harvested RJ (4.77 0.25%), which was successively produced by worker bees without resting rotation (p < 0.05). The amount of 10-HDA in these RJ samples were also compared in **Figure 11** and was also found to decrease in 72-h-harvested RJ without resting rotation (48-h-harvested RJ, 2.9 0.2%; 72-h-harvested RJ, 2.5 0.1%; p < 0.05). The author has been emphasized in his proposal on natural beekeeping that health of worker bees should be guaranteed by rotation of beehives employed for RJ production, as well as by limiting number of artificial queen cells per colony. The results also support his proposal for production of high-quality RJ.

### *3.4.2 Reasons why 10-HDA and MRJP-1 multimer are suitable to quality assessment of RJ*

10-HDA is the only functional component used for the quality control of RJ (**Table 2**). The author strongly agrees that 10-HDA should be used as the quality control of RJ. Unfortunately, since authentic compound is available and easily added to the product, 10-HDA does not play an important role in the evaluation of RJ. Thus, I proposed one more functional component, MRJP-1 multimer, because this is also a compound only produced by honeybees [28, 30] and it too requires exacting conditions in order to maintain freshness. MRJP-1 is found in a large amount, ca. 60% of soluble protein in RJ, which makes it easy to use as a determining factor. MRJP-1 multimer decomposes easily at high temperature, such as 30°C and higher, over a period of 2 weeks (**Figure 12**) [26].

The transitional change of MRJP-1 multimer contents stored under different temperatures is shown in **Figure 12**. The MRJP-1 multimer contents decreased depending on temperature and period of storage, with a reduction of about 50% in RJ

over quality and seeks cheapness with a production-first policy and cost-first policy in pursuit of mass production; and is causing weakened colonies and shortened lifespans through an artificial mating process called "selective breeding". Especially in China, the honeybees dedicated for royal jelly production tend to be used after "selective breeding" and even the 10-HDA content has been decreasing in recent times. In this context, it is apparent that society's confidence in apicultural products including royal jelly will soon be lost and the apicultural industry will enter a course

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

Accordingly, I have performed research and practices to solve the various problems for the purpose of contributing to development of the apicultural industry. As a result, I have reached the conclusion that only natural beekeeping producing true apicultural products can protect the quality of apicultural products including royal jelly and can also contribute to the health of humankind. It is needless to say that such natural beekeeping will eventually also improve beekeepers'standards of living.

I have practiced natural beekeeping based on the original ecology of honeybees in Qing hai Menyuan, China over 20 years since 1993. It is necessary to request beekeepers' cooperation for production of excellent royal jelly of a high quality. Especially when harvesting RJ after 48 h, the frequency of harvest operation is increased from once every 3 days to once every 2 days in comparison with harvesting after 72 h. Furthermore, the amount produced when harvesting after 48 h is half of the amount produced when harvesting after 72 h. In other words, the method proposed by me leads to intensification of labor and decreased production efficiency. In addition, in conventional beekeeping, 200–300 artificial queen cell cups are set in one comb for RJ secretion, while in natural beekeeping the number of artificial queen cell cups is limited to not more than 100. The Natural Beekeeping therefore met with stiff opposition due to its resultant low production efficiency. Nevertheless, the author persuaded the beekeepers with a passion for production of the world's leading apicultural products. The philosophy of "high quality, high price" was explained to the beekeepers. Production was encouraged with the basic principle that "only high-quality products can be sold at high prices", and as a result, natural beekeeping has been able to give plenitude to beekeepers' lives and contribute to the improvement of their standards of living. This achievement is of

Looking at the state of RJ production in 2011, the abnormally dry weather was influential, but the high rate of inflation in China was more influential. Migratory beekeepers have to carry their beehives to distant bee forage areas by chartering trucks, while there was also a steep increase in transportation fees. Furthermore, larva transfer is an important job in RJ production. Since it is difficult for the old beekeepers themselves to perform these operations, young persons are hired and instructed regarding how to perform the operations in the dark under a tent. Beekeepers should be able to live comfortably by realizing the true philosophy of "high quality, high price". Otherwise, it is feared that the apicultural industry will

The growth of larvae cultured in RJ on 3 and 6 days after the cultivation is shown in **Figure 13** [26]. The graph shows the results with RJ harvested at 48 and 72 h after transfer of queen cells. It was found that the weight of larvae had increased rapidly by more than seven times within 3 days. The increase of body weight, however,

became slowed to a factor of only 1.2–1.4 over the following 3 days.

of decline.

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

great significance.

enter a course of decline.

**3.5 Biological effect of RJ**

**141**

*3.5.1 Effect of RJ on growth of larvae*

**Figure 12.** *Transitional change of MRJP-1 multimer contents stored under different temperature [26].*

samples stored at 35°C for 90 days and as much as an 80% reduction after storage at 40°C for 90 days. Furusawa et al. [31], also reported that apisin (MRJP-1 multimer) content in RJ was also decreased by approximately 80% at 40°C after 32 days.

Kamakura et al. also proposed 57-kDa protein in RJ as a possible marker for freshness [29]. MRJP-1 monomer has molecular weight of 55 kDa and is the component of MRJP-1 multimer (pentamer or hexamer depending upon the report). Kamakura reported that 57-kDa protein is identical to MRJP-1 monomer.

Among the proteins found in RJ, including MRJP-1 to MRJP-9 and other specific proteins, MRJP-1 multimer and MRJP-1 monomer were the compounds capable of bearing thermolability. Li et al. reported that quantity of MRJP-1 decreased significantly following the temperature trend in 2D-PAGE, MALDI TOF/TOF MS images, but MRJP-2 and MRJP-3 did not increase or decrease following the temperature trend [32]. However, MRJP-4, 5, glucose oxidase, peroxiredoxin, and glutathione S-transferase S1 were clearly absent in all images in samples held at room temperature for 1 year. I indicated that as MRJP-1 multimer was unstable at room temperature within short period, MRJP-1 multimer might be the substance for use as the possible marker for freshness. Although MRJP-4 and MRJP-5 should be studied further, I believe that MRJP-1 multimer is the compound that should be used as the marker for the quality control. Takenaka et al. also reported that the lowering of glucose oxidase activity was found and almost disappeared within 120 days at room temperature in the dark. However, the amount of 10-HDA, 10-hydroxydecanoic acid, and gluconic acid in RJ were constant regardless of the storage condition [33]. The author found that MRJP-1 multimer was stable at 2°C or 18°C. According to the most recent findings, the core structure of the MRJP1 multimer consists of four molecules of MRJP1, four molecules of the peptide apisimin [34] and, surprisingly, eight molecules of 24-methylenecholesterol [35, 36].

Since MRJP-1 multimer or MRJP-1 monomer cannot be chemically synthesized, and MRJP-1 monomer is unstable at even at room temperature, MRJP-1 multimer is exactly the right compound for the quality control of RJ in addition to 10-HDA [37].

#### *3.4.3 Significance of production of natural RJ utilizing artificial queen cell cups*

As mentioned above in detail, the modern beekeeping industry ignores the highlevel biological functions possessed originally by honeybees; emphasizes quantity

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

over quality and seeks cheapness with a production-first policy and cost-first policy in pursuit of mass production; and is causing weakened colonies and shortened lifespans through an artificial mating process called "selective breeding". Especially in China, the honeybees dedicated for royal jelly production tend to be used after "selective breeding" and even the 10-HDA content has been decreasing in recent times. In this context, it is apparent that society's confidence in apicultural products including royal jelly will soon be lost and the apicultural industry will enter a course of decline.

Accordingly, I have performed research and practices to solve the various problems for the purpose of contributing to development of the apicultural industry. As a result, I have reached the conclusion that only natural beekeeping producing true apicultural products can protect the quality of apicultural products including royal jelly and can also contribute to the health of humankind. It is needless to say that such natural beekeeping will eventually also improve beekeepers'standards of living.

I have practiced natural beekeeping based on the original ecology of honeybees in Qing hai Menyuan, China over 20 years since 1993. It is necessary to request beekeepers' cooperation for production of excellent royal jelly of a high quality. Especially when harvesting RJ after 48 h, the frequency of harvest operation is increased from once every 3 days to once every 2 days in comparison with harvesting after 72 h. Furthermore, the amount produced when harvesting after 48 h is half of the amount produced when harvesting after 72 h. In other words, the method proposed by me leads to intensification of labor and decreased production efficiency. In addition, in conventional beekeeping, 200–300 artificial queen cell cups are set in one comb for RJ secretion, while in natural beekeeping the number of artificial queen cell cups is limited to not more than 100. The Natural Beekeeping therefore met with stiff opposition due to its resultant low production efficiency. Nevertheless, the author persuaded the beekeepers with a passion for production of the world's leading apicultural products. The philosophy of "high quality, high price" was explained to the beekeepers. Production was encouraged with the basic principle that "only high-quality products can be sold at high prices", and as a result, natural beekeeping has been able to give plenitude to beekeepers' lives and contribute to the improvement of their standards of living. This achievement is of great significance.

Looking at the state of RJ production in 2011, the abnormally dry weather was influential, but the high rate of inflation in China was more influential. Migratory beekeepers have to carry their beehives to distant bee forage areas by chartering trucks, while there was also a steep increase in transportation fees. Furthermore, larva transfer is an important job in RJ production. Since it is difficult for the old beekeepers themselves to perform these operations, young persons are hired and instructed regarding how to perform the operations in the dark under a tent. Beekeepers should be able to live comfortably by realizing the true philosophy of "high quality, high price". Otherwise, it is feared that the apicultural industry will enter a course of decline.

### **3.5 Biological effect of RJ**

### *3.5.1 Effect of RJ on growth of larvae*

The growth of larvae cultured in RJ on 3 and 6 days after the cultivation is shown in **Figure 13** [26]. The graph shows the results with RJ harvested at 48 and 72 h after transfer of queen cells. It was found that the weight of larvae had increased rapidly by more than seven times within 3 days. The increase of body weight, however, became slowed to a factor of only 1.2–1.4 over the following 3 days.

samples stored at 35°C for 90 days and as much as an 80% reduction after storage at 40°C for 90 days. Furusawa et al. [31], also reported that apisin (MRJP-1 multimer) content in RJ was also decreased by approximately 80% at 40°C after 32 days. Kamakura et al. also proposed 57-kDa protein in RJ as a possible marker for freshness [29]. MRJP-1 monomer has molecular weight of 55 kDa and is the component of MRJP-1 multimer (pentamer or hexamer depending upon the report).

Among the proteins found in RJ, including MRJP-1 to MRJP-9 and other specific proteins, MRJP-1 multimer and MRJP-1 monomer were the compounds capable of bearing thermolability. Li et al. reported that quantity of MRJP-1 decreased significantly following the temperature trend in 2D-PAGE, MALDI TOF/TOF MS images, but MRJP-2 and MRJP-3 did not increase or decrease following the temperature trend [32]. However, MRJP-4, 5, glucose oxidase, peroxiredoxin, and glutathione S-transferase S1 were clearly absent in all images in samples held at room temperature for 1 year. I indicated that as MRJP-1 multimer was unstable at room temperature within short period, MRJP-1 multimer might be the substance for use as the possible marker for freshness. Although MRJP-4 and MRJP-5 should be studied further, I believe that MRJP-1 multimer is the compound that should be used as the marker for the quality control. Takenaka et al. also reported that the lowering of glucose oxidase activity was found and almost disappeared within 120 days at room temperature in the dark. However, the amount of 10-HDA, 10-hydroxydecanoic acid, and gluconic acid in RJ were constant regardless of the storage condition [33]. The author found that MRJP-1 multimer was stable at 2°C or 18°C. According to the most recent findings, the core structure of the MRJP1 multimer consists of four molecules of MRJP1, four molecules of the peptide apisimin [34] and, surprisingly,

Since MRJP-1 multimer or MRJP-1 monomer cannot be chemically synthesized, and MRJP-1 monomer is unstable at even at room temperature, MRJP-1 multimer is exactly the right compound for the quality control of RJ in addition to 10-HDA [37].

As mentioned above in detail, the modern beekeeping industry ignores the highlevel biological functions possessed originally by honeybees; emphasizes quantity

*3.4.3 Significance of production of natural RJ utilizing artificial queen cell cups*

Kamakura reported that 57-kDa protein is identical to MRJP-1 monomer.

*Transitional change of MRJP-1 multimer contents stored under different temperature [26].*

*Modern Beekeeping - Bases for Sustainable Production*

**Figure 12.**

**140**

eight molecules of 24-methylenecholesterol [35, 36].

#### **Figure 13.**

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

The growth of larvae at Day 0, Day 3 and Day 6 is also represented in **Figure 14**. It was also confirmed that the larvae grew rapidly in the RJ in vitro [26].
