**3.4 Major components of RJ obtained by natural beekeeping**

**Table 2** shows the standard values of major components of RJ proposed by the associations of China, the world's biggest apicultural product-manufacturing country, and Japan. Among the major components for which standard values are specified, 10-HDA is clearly the characteristic fatty acid contained in RJ. However, it is problematic that the quality of RJ is assessed only based on 10-HDA even in the case of poor storage conditions, since 10-HDA is relatively stable regardless of heat and can intentionally be added at a later point in time. I would like to emphasize that MRJP-1 multimer, which is the most abundant protein found in RJ, should be adopted as a new index to assess the quality of RJ in addition to 10-HDA. The reasons for this are that MRJP-1 multimer accounts for the major part (40–60%) of water-soluble proteins (about 75% of soluble proteins) among the entire range of proteins (11–15 g/100 g RJ), it cannot be added artificially, and it is easily decomposed by heating. The RJ obtained by Natural Beekeeping is compared below with that obtained by ordinal beekeeping to show that the former is superior in quality and can maintain the strength of the colony.

## *3.4.1 Comparison of contents of MRJP-1 Multimer and 10-HDA in RJ between natural beekeeping and ordinal beekeeping*

I compared the component contents between the RJ obtained by Natural Beekeeping (performed in the area with good beekeeping environments (Qing Hai Haibei Menyuan, harvested after 48 h) and that obtained by ordinal beekeeping (harvested after 72 h)). This experiment used colonies from which no RJ had been harvested within 1 month before the start of the experiment. After starting the experiment, RJ was harvested by Natural Beekeeping (after 48 h) and ordinal beekeeping (after 72 h).

#### *3.4.1.1 Harvest of RJ*

The RJ was harvested according to the method of Natural Beekeeping proposed by me [5, 6]. Basically RJ was prepared in the following manner.

The core of the Natural Beekeeping is respect for the ecology and providence of honeybees. Based on this fundamental recognition, the rearing environments and facilities are arranged in such a way that keeps honeybees healthy and vigorous at all occasions, and the honeybee-rearing methods and apicultural product-manufacturing methods to create RJ and honey of the highest quality are practiced.

The concept of natural beekeeping is as follows: "Specific beekeeping where the nectar plants are natural plants growing wild, the ecology and providence of honeybees are respected and harsh harvesting is not adopted. In addition, no artificial foods are given, and any drugs including antibiotics are not used. Instead, the health of honeybees is controlled with good environments and good rearing management."

The details of the Natural Beekeeping are outlined below:

1.The ecology and providence of honeybees and their society are respected.

2.Honeybees are not abused.

3.Honeybees are fed with natural honey and pollen, and are not given artificial pollen made from sugar water or soybeans.

**Item**

**133**

Description

 A yellowish white milky liquid substance with a specific odor,

a weak acidic taste and an

astringent property

**Natural Beekeeping**

**Specifications1**

 **RJ**

**Food Standards and Criteria**

**Standards of Japan Royal**

**National Standards of**

**ISO 128245**

> **People's Republic of China4**

**Color tone**: A milky white, pale

Royal jelly is milky white, pale yellow, white luster. It is pasty

yellow or pale orange color

surely

When frozen, there must be

also a cryohydric gloss. **Odor:** In a creamy state, there

must be an odor like flower

nectar or pollen and an

acrimonious

must be pure, and there must

not be a foul odor.

**Taste and texture:** In a creamy

state, there are a clear acidic

taste, a bitter taste, an

acrimonious

taste, and the maxilla and

throat feel stimulation.

swallowed or spit out,

stimulation

throat for a certain period of

time. In a frozen state, there is a

granular feeling after put into the mouth, but

such feeling disappear gradually and the same tastes

are felt as in the creamy state.

**State:** The creamy royal jelly at

ordinary temperature

thaw has fluidity. It must not be

contaminated

matters such as bubbles or wax

scum.

 with foreign

 or after

immediately

 remains at the

 When

 taste and a sweet

fermentation

 odor or a

 odor. The odor

accompanied

 by a gloss.

or jelly-like at room

temperature

shall be free from bubbles and

foreign substances.

crystallization

occur naturally in royal jelly

during storage. Odor and Taste: It is pungent,

unfermented

rancescent.

and it brings acrid taste to

palate and throat.

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

 It is acerb, spicy

 and shall not be

 phenomena

 can

 Minor

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

 with fluidity and

**Jelly Fair Trade Council3**

Generally, a milky white or

light yellow paste-like substance with a specific astringent property and a

flavor

**for RJ2** A yellowish white milky liquid substance with a specific odor,

a weak acidic taste and an

astringent property


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

**3.4 Major components of RJ obtained by natural beekeeping**

*Modern Beekeeping - Bases for Sustainable Production*

**Table 2** shows the standard values of major components of RJ proposed by the associations of China, the world's biggest apicultural product-manufacturing country, and Japan. Among the major components for which standard values are specified, 10-HDA is clearly the characteristic fatty acid contained in RJ. However, it is problematic that the quality of RJ is assessed only based on 10-HDA even in the case of poor storage conditions, since 10-HDA is relatively stable regardless of heat and can intentionally be added at a later point in time. I would like to emphasize that MRJP-1 multimer, which is the most abundant protein found in RJ, should be adopted as a new index to assess the quality of RJ in addition to 10-HDA. The reasons for this are that MRJP-1 multimer accounts for the major part (40–60%) of water-soluble proteins (about 75% of soluble proteins) among the entire range of

proteins (11–15 g/100 g RJ), it cannot be added artificially, and it is easily

quality and can maintain the strength of the colony.

*beekeeping and ordinal beekeeping*

beekeeping (after 72 h).

*3.4.1.1 Harvest of RJ*

practiced.

**132**

decomposed by heating. The RJ obtained by Natural Beekeeping is compared below with that obtained by ordinal beekeeping to show that the former is superior in

*3.4.1 Comparison of contents of MRJP-1 Multimer and 10-HDA in RJ between natural*

I compared the component contents between the RJ obtained by Natural Beekeeping (performed in the area with good beekeeping environments (Qing Hai Haibei Menyuan, harvested after 48 h) and that obtained by ordinal beekeeping (harvested after 72 h)). This experiment used colonies from which no RJ had been harvested within 1 month before the start of the experiment. After starting the experiment, RJ was harvested by Natural Beekeeping (after 48 h) and ordinal

The RJ was harvested according to the method of Natural Beekeeping proposed

The concept of natural beekeeping is as follows: "Specific beekeeping where the nectar plants are natural plants growing wild, the ecology and providence of honeybees are respected and harsh harvesting is not adopted. In addition, no artificial foods are given, and any drugs including antibiotics are not used. Instead, the health of honeybees is controlled with good environments and good rearing management."

1.The ecology and providence of honeybees and their society are respected.

3.Honeybees are fed with natural honey and pollen, and are not given artificial

The core of the Natural Beekeeping is respect for the ecology and providence of honeybees. Based on this fundamental recognition, the rearing environments and facilities are arranged in such a way that keeps honeybees healthy and vigorous at all occasions, and the honeybee-rearing methods and apicultural product-manufacturing methods to create RJ and honey of the highest quality are

by me [5, 6]. Basically RJ was prepared in the following manner.

The details of the Natural Beekeeping are outlined below:

pollen made from sugar water or soybeans.

2.Honeybees are not abused.


**Item**

**135**

Ash Starch Total lipid C13/C12 Isotopic

ratio(δ%)

Viable microbe

Not more than 500 cfu/g

 Not more than 500 cfu/g

 Not more than 500 cfu/g

count

*E. coli* Enterobacteriaceae

Salmonella

Heavy metals

Arsenic Residual

HC, DDT or dieldrin family

must not be detected.

agrochemical

Antibiotic

Tetracycline

 family,

chloramphenicol

streptomycin

detected.

*1Specifications*

*2Japan Health and Nutrition Food Association.*

*3From* *4From* *5ISO 12824* 

**Table 2.** *Quality standards and criteria for native RJ.*

*"GB/T9697-2002*

*(2016/09/15).*

*Amendment".*

*"Practice Rules for Fair Competition*

 *Code Related to Display of Royal Jelly" of Japan Royal Jelly Fair Trade Council.*

 *of royal jelly harvested by the Natural Beekeeping (acceptance*

 must not be

 or

Not more than 2 ppm as As

 Not more than 2 ppm as As

HC, DDT or dieldrin family

must not be detected.

Tetracycline

 family,

chloramphenicol

streptomycin

detected.

 *criteria).*

 must not be

 or

 Not more than 20 ppm as Pb Not more than 20 ppm as Pb

Negative

Negative

Negative

**Natural Beekeeping**

**Specifications1**

 **RJ**

**Food Standards and Criteria**

**Standards of Japan Royal**

**National Standards of**

**ISO 128245**

> **People's Republic of China4**

Not more than 1.5%

No detection 2–8%

Type 1:

Type 2:

Max: 500 Absent in 10 g Absent in 25 g

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

29 to

14

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

29 to

20

**Jelly Fair Trade Council3**

**for RJ2**


**Table 2.** *Quality standards and criteria for native RJ.*

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

**Item**

**134**

Water content

Crude protein

**10-HDA** **MRJP-1 multimer**

Acidity

 **Not less than 5.0%** Not less than 32 mL and not

Not less than 32 mL and not

Not less than 32.0 mL and not

Not less than 30 mL and not

Min: 30.0 mL

Max: 53.0 mL

more than 53 mL of 1 mol/L

NaOH for 100 g of royal jelly

Not more than 15%

Min: 7% Max: 18%

2–9%

2–9%

Type 1: <3.0%

Type 2: Na6

Type 1: <0.5%

Type 2: Na

Type 1: <1.5%

Type 2: Na

Type 1: <0.5%

Type 2: Na

more than 53.0 mL of 1 mol/L

NaOH for 100 g of royal jelly

more than 53 mL of 1 mol/L

NaOH for 100 g of royal jelly

more than 53 mL of 1 mol/L

NaOH for 100 g of royal jelly

> Total sugar

Fructose

Glucose Sucrose

Erose Maltose Maltotriose

**Not less than 2.0%**

 Not less than 11.0% and not

Not less than 11.0% and not

more than 14.5%

**Not less than 1.6%**

more than 15.5%

 Not less than 62.5% and not

more than 68.0%

**Natural Beekeeping**

**Specifications1**

 **RJ**

**Food Standards and Criteria**

**Standards of Japan Royal**

**National Standards of**

**ISO 128245**

> **People's Republic of China4**

(Excellent product) Not more

Min: 62.0%

Max: 68.5%

than 67.5% (Acceptable

than 69.0%

 product) Not more

**Jelly Fair Trade Council3**

Not less than 62.5% and not

more than 68.5%

(Acceptance

than 63.0% and not more than

68.0%)

Not less than 12.0% and not

Not less than 11% and not more

Min: 11% Max: 18%

*Modern Beekeeping - Bases for Sustainable Production*

**Min: 1.4%**

than 16% **(Excellent product) Not less**

**than 1.8%** **(Acceptable**

**than 1.4%**

 **product) Not less**

more than 15.5%

**Not less than 1.4%**

**(Acceptance**

**less than 1.6%)**

 **criterion: Not**

 criterion: Not less

**for RJ2**

Not less than 62.5% and not

more than 68.0%


*3.4.1.2.1 Preparation of sample solution*

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

filtrate was used as the sample solution.

*3.4.1.2.2 Preparation of standard solution*

used as the standard solution.

*3.4.1.2.3 Quantitative analysis*

the following formula.

(1:1).

**137**

14–16 min.

After homogenizing the sample RJ, 0.5 g of RJ was weighed accurately into a 200 ml beaker. Water 80 ml was added and the mixture was stirred vigorously until a uniform suspension was obtained. Next, methanol 80 ml was added, and the mixture was further stirred for 20 min and transferred into a 200 ml measuring flask. The beaker was washed with a mixture of water and methanol (1:1), and the washing was added to the 200-ml measuring flask. The mixture of water and methanol (1:1) was added to the measuring flask to make exactly 200 ml. The resulting fluid was filtered with a membrane filter 0.45 μm in pore size, and the

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

Exactly 0.01 g of 10-HDA Reference Standard was weighed accurately and dissolved in methanol to make 50 ml. Exactly 5 ml of this solution was added with the mixture of water and methanol (1:1) to make 20 ml. The resulting solution was

The test was performed with 10 μl each of the sample solution and the standard solution as directed under the Liquid Chromatography in line with the following conditions. The areas of 10-HDA peaks from both solutions, At and As, were determined, and the amount of 10-HDA in the sample was calculated according to

¼ weighed amount mg ð Þ of 10 � HDA Reference Standard � At*=*As

where As is the area of 10-HDA peak from the standard solution and At is the

Mobile phase: A mixture of diluted phosphoric acid (1 in 1000) and methanol

Flow rate: Adjusted so that the retention time of 10-HDA was in the range of

MRJP-1 is a major protein of RJ. It is a multifunctional pharmaceutically impor-

tant protein [24–26] and serves as a marker of the authenticity and quality of honeybee products [27, 28]. The analysis of MRJP-1 multimer in RJ was performed

Reproducibility: When the test was repeated six times with the standard solution in line with the above conditions described above, the relative standard

Detector: An ultraviolet absorption photometer (wavelength: 210 nm).

Column temperature: A constant temperature of about 40°C.

deviation of 10-HDA peak areas was not more than 2.0%.

by the method reported previously (partially modified) [29].

Amount mg ð Þ of 10 � HDA in 100 g of sample

Column: An ODS column (4.6 mm<sup>Φ</sup> � 150 mm).

area of 10-HDA peak from the sample solution.

*3.4.1.2.4 HPLC operating conditions*

*3.4.1.3 Analysis of MRJP-1 Multimer in RJ*

� 100 g*=*weighed amount gð Þ of sample


10.Both honey and RJ are always filtered at the apiary.


In Qing hai Haibei Menyuan, RJ was harvested from rape blossoms (almost in full bloom) using Occidental honeybees (*Apis mellifera*) for 15 days from July 10–24, 2010.

For each sample, 60 g was harvested as follows:

