Effect of varying amounts of burnt Boswellia Frankincense stick gases on refractive index (%) of sugar in sapodilla

How does the amount of Boswellia serrata burnt (0g, 5g, 10g, 15g, 20g, 25g) affect the refractive index (%) of sugar and the mass of Manilkara zapota (chikoo)  at room temperature?

I had taken a road trip to rural Maharashtra, a journey filled with hills and farmland. We stopped at one of the farms, to buy fresh produce, and I got to speaking with the farmer, who told me that vendors use agarbati (Indian frankincense sticks) to ripen produce. This peaked my interest, as I found it unusual. After some reading, I came across a few articles which supported the farmer’s claim. When fruits ripen organically, they release ethylene gas, which is proven to improve their ripening(India). As ethylene is a natural hormone, it does not cause any harm to the health of the consumers of the fruit. With further research I discovered that ethylene was released by the burning of Indian frankincense (Boswellia serrata), which is the main ingredient of agarbatti, also known as loban (Nyanjage et al.) Hence, I started to research on the differences between the natural and artificial process of ripening.  My research also led me to several articles on benefits and disadvantages of artificial ripening.  It led me to ask questions like what exactly happens in the process of fruit ripening and whether we can ensure exactly the right level of fruit ripening for human consumption.

Ripening is a natural physiological process that makes the fruit sweeter, more palatable, edible, nutritious, softer and attractive. Ripening is also associated with color change due to the pigments that are already present or are produced during ripening (Vaviya et al.) If the ripening process is allowed to continue, the fruit reaches senescence i.e. it becomes overly ripe and now it’s only aim is seed dispersal. It is a natural process and can be slowed down or speeded up, but cannot be completely inhibited. Ethylene gas helps in this process.

Incense sticks produce Ethylene gas on burning. (Marrero et al.) Ethylene is also a gaseous phytohormone produced by plants to carry out both processes of growth and senescence.  It is released naturally to promote fruit ripening (Iqbal et al.). The ripening of fruits is a unique coordination of various biochemical and developmental pathways regulated by ethylene, which affects color, texture, nutritional quality and aroma of fruits. During ripening in climacteric fruits, the ethylene regulates firmness and color changes involving chlorophyll reduction, increase in carotenoids or anthocyanins, sugars, and biosynthesis of volatile organic compounds (Iqbal et al., “Ethylene Role in Plant Growth, Development and Senescence”). Hence it can be theorized that incense sticks will promote artificial ripening of fruits due to presence of ethylene.

In optics, the refractive index or index of refraction n of a material is a dimensionless number that describes how light propagates through that medium. It is defined as:

n = c/v

where, c is the speed of light in vacuum and v is the phase velocity of light in the medium. For example, the refractive index of water is 1.333, meaning that light travels 1.333 times faster in a vacuum than it does in water.

The refractive index of a liquid can be measured using a hand refractometer ( a handheld device that works on the critical angle principle by which lenses and prisms project a shadow line onto a small glass reticle inside the instrument, which is then viewed by the user through a magnifying eyepiece. It can be digital or manual) As the concentration of dissolved solids in the liquid changes, the refractive index changes. Increasing concentration of dissolved solids increases the amount of refraction while the amount of refraction decreases with decrease in dissolved solids. In case of increase in process of fruit ripening, an increase in the concentration of sugars leads to an increase in the percentage refractive index. It is this property which is used in many food industries for checking the quality of food products. We shall be using this property for checking amount of sugar dissolved in the extract. (Contento et al.)

According to research, a fruit will increase in mass as long as sugar is still being transported into it by the plant.  This is possible only as long as the fruit is attached to the main plant i.e. prior to harvesting.  A climacteric fruit is one which can be harvested raw and then allowed to ripen.  This increases the shelf life of the fruit. However, once the fruit is harvested, no further sugar reserves enter the fruit and the change in mass is generally shown as a decline in mass. (Prevention of Post-Harvest Food Losses Fruits.)

The decrease in weight of the climacteric fruits on ripening and its ripening is because of three reasons (a) loss of water due to evaporation from surface (b) life processes like respiration being continuously carried out in the plant which reduces the dry matter content in fruit for energy  and (c) hydrolysis of starch into sugar by amylase enzyme.  The greater the decrease in mass, the greater is the ripening of the fruit.

• The percentage refractive index of sugar in Manilkara zapota extract will increase as the amount of Boswellia serrata burnt increases at room temperature.

This is reasoned as ethylene gas is released by burning Boswellia serrata which is a gaseous hormone that hastens ripening in fruits.

• The mass of the fruit (Manilkara zapota) extract will decrease as the amount of Boswellia serrata burnt increases at room temperature.

This is reasoned as the fruit has already been harvested and the biochemical changes and water loss can cause reduction in mass.

• The percentage of refractive index of sugar in Manilkara zapota extract will not show any significant difference as the amount of Boswellia serrata burnt increases at room temperature.
• 2) The mass of the fruit (Manilkara zapota) extract will not show any significant difference as the amount of Boswellia serrata burnt increases at room temperature.

The main steps for conducting this study were:

• Procurement of raw sapodilla and incense sticks from the market
• Carry out ripening in controlled conditions for  a period of 5 days
• Extract samples from each of the ripened fruits.
• Measure refractive index of each sample using the refractometer.
• Comparison of the amount of sugar present in test samples with respect to control samples of sapodilla (where no incense sticks were used to influence the ripening process).
• Processing of raw data  and its statistical analysis.

There were no ethical concerns in this project.

Procedure:-

• Procuring raw sapodilla was done from a farm locally.
• The Boswellia serrata was prepared in the following manner.
  • Boswellia serrata sticks were crushed in the mortar and pestle
  • A piece of lab paper was  placed onto the weighing scale and then  the scale was tared (reset to zero).
  • Using the spatula, 5.0 g of powdered Boswellia serrata was measured and wrapped in the lab paper itself.
  • Similar packets were created for 10.0 g, 15.0 g, 20.0 g, and 25.0 g of powdered Boswellia serrata and lab paper was wrapped around the Boswellia serrata to secure it.
• Six boxes were prepared in the following manner:
  • The boxes were numbered from 1-6. The first box did not contain any Boswellia serrata. Boxes 2-6  contained 5.0 g, 10.0 g, 15.0 g, 20.0 g, and 25.0 g of Boswellia serrata respectively.
  • Five  raw fruits were weighed first individually on the mass balance.
  • They were then put into a box, ensuring that they were not touching each other.
  • The measured amount of Boswellia serrata was placed into a steel bowl and the bowl was placed in the box, next to the fruits. This step applied to all boxes except Box 1.
  • Using a matchstick, the Boswellia serrata was lighted on fire and the container’s lid was halfway covered.
  • Once the Boswellia serrata had finished burning, the box was closed and taped shut.
  • Let sit for 5 days.
• At the end of the said period, the following procedure was followed for contents of Box 1.
  • Each fruit was weighed individually and the final weights noted.
  • Each fruit was then chopped separately and placed in separate bowls.
  • The chopped pieces from one bowl were pureed using a blender.
  • Filter paper was first lined on the funnel and the funnel was placed above a beaker.  The puree was poured on the funnel and the fruit juice (minus the fibres) was collected in the beaker.
  • This was done for all five fruits and the juices were transferred into 5 different test tubes.
  • Using a dropper,  a drop of the juice from one fruit was placed onto the face of the hand refractometer.
  • The measurement was taken and repeated for all five fruits as five trials.
• The above steps were repeated for  Boxes 2-6.
• Raw Data was processed and appropriate conclusions were drawn using statistical analysis.

Formula used: Change in mass (m ± 0.2 g) = ( m₁ ± 0.1g) - (m₂ ± 0.1 g)

Average increase in mass = ( Trial-1+Trial-2+Trial-3+Trial-4+Trial-5)/ 5

Standard Deviation \(=\ \sqrt{\frac{\sum(x\ -\ ẋ)^2}{n\ -\ 1}}\)  where x = increase in mass, ẋ = average mass increase,n = number of trials

From the trendline in Figure 16, it shows that there is a negative correlation between the average decrease in mass of fruit on ripening and the amount of incense burnt. This is because we can see that the trendline is moving downhill.  However if we study the individual values, all values showing presence of incense sticks show an uphill trend as for eg, at 5g dhoop burning,  average difference in fruit mass is 6.42g, while for 15 g dhoop, it is 6.72 g.  Then at 25 g dhoop, it is 7.08 g.  There is a possibility that data of the control is anomalous and may need to be reconsidered.

As the fruit is getting ripened post harvest (i.e. it is no longer attached to the tree where it grew) there is no fresh inflow of soluble material.  At the same time there are biochemical changes occurring in the fruit and also evaporation is taking place continuously reducing its water content.  The mass gets decreased at the end of the experimental period.

Average refractive index = ( Trial-1 + Trial-2 + Trial-3 + Trial-4 + Trial-5)/ 5

Standard Deviation \(=\ \sqrt{\frac{\sum(x\ -\ ẋ)^2}{n\ -\ 1}}\) where x = refractive index reading, ẋ = average refractive index, n = number of trials

From the trendline, which is going uphill from left to right, we can say that there is a positive correlation between the average refractive index of the ripened fruit and the amount of incense sticks burnt for the ripening process. For example, when the amount of dhoop burnt is 5 g, the refractive index is 16.6%, which increases to 18% and 21 % when the amount of burnt incense becomes 15g and 25g.  As fruit gets ripened, it becomes sweeter due to greater conversion of carbohydrates into sugars.  As the number of dissolved sugar molecules increases in the fruit extract, the refractometer which is calibrated with greatest transparency (RO water) at 0%, will show a steady increase in its percentage reading due to reduction in transparency.

The purpose of this t-test of independence is to understand if there is any significant difference between the way the mass of  Boswellia serrata burnt impacts the decrease in mass of the fruit and the refractive index in %.

H_o: The change in mass of fruit and the refractive index in % has no significant correlation between them.

H₁: The change in mass of fruit and the refractive index in % has significant correlation between them.

Level of confidence (α) = 0.05

As there are 6 variables(n = 6), the degrees of freedom is (6-1) = 5.

The test is done at a significance level of 0.05.

The critical value at this level is 0.727

The t value is calculated as shown:

\(\frac{(\sum(x-y))/n}{\sqrt\frac{\sum(x-y)^2}{(n-1)n}-\frac{(\sum(x-y))^2}{n}}\) = 0.42

The value of t is 0.42

The calculated t value is 0.42 which is lower than the critical value of 0.727. This allows us to accept the null hypothesis and reject  the alternate one.

Hence the experimental hypothesis is the change in mass of fruit and the refractive index in % has no significant correlation between them.

Experimental Hypothesis

• The percentage refractive index of sugar in Manilkara zapota extract will increase as the amount of Boswellia serrata burnt increases at room temperature.
• The mass of the fruit (Manilkara zapota) extract will decrease as the amount of Boswellia serrata burnt increases at room temperature.

The purpose behind literature reference is to find out how much research has been carried out on the selected research topic and how I could improve on my methodology and analysis with the help of other researchers.  For this, I looked up various academic papers submitted on related topics. A short review is provided. I learnt that there are various changes that occur during the ripening process in papaya. Sucrose synthesis in the ripening process does not stop even post harvest and as the fruit ripens, the pulp softening leads to easier release of sucrose from the cells, thus increasing the sweetness index. (Gomez, M. L. P. A. et al., 2002) The sapodilla fruit goes through a similar ripening process.  I got to know that sapodilla has a very short shelf life due to the fast ripening process.  I now wanted to confirm from my experimental work, that it was indeed so. (Moo-Huchin, V. M. et al., 2013). I also read several papers on different methods to do a quantitative analysis on a test for detection of carbohydrates.  I got to know about the DNS method of quantifying results of tests for sugars.  However, since the chemicals involved were very toxic, I did not consider this method. (Garriga, M. et al., 2017). I learnt about the use of a software called MATLAB that can potentially be used to distinguish between naturally and artificially ripened fruits. (Hatmore, N. et al., 2020). There are several materials and methods to extract and purify water soluble polysaccharides in fruits. (John, A. et al., 2018)

Research Question: How does the amount of Boswellia serrata burnt (0g, 5g, 10g, 15g, 20g, 25g) affect the refractive index (%) of sugar and the mass of Manilkara zapota (chikoo)  at room temperature?

The experimental data analysis shows that there is a positive correlation between percentage refractive index and the mass of Boswellia serrata burnt If we study the individual values, all values showing presence of incense sticks show an uphill trend as for eg, at 5g dhoop average difference in fruit mass is 6.42g, while for 15 g dhoop, it is 6.72 g.  Then at 25 g dhoop, it is 7.08 g.   This means that the more the amount of incense used, the greater is the value of the percentage refractive index. This is because the amount of sugar increases with increase in fruit ripening and thus gives a greater refractive index reading.  This means that as the amount of incense burnt increases, ripening also increases.

The experimental data analysis shows that there is a positive correlation between decrease in fruit mass and the mass of      Boswellia serrata burnt.  For example, ]-when the amount of dhoop burnt is 5 g, the refractive index is 16.6%, which increases to 18% and 21 % when the amount of burnt incense becomes 15g and 25g.This means that the more the amount of incense used, the greater is the decrease in mass of the fruit. This is because the amount of sugar increases with increase in fruit ripening, as does the level of dehydration.  This means that as the amount of incense burnt increases, ripening also increases.

The above analysis is supported by the visual observations also. The initial readings of control show only a mild level of softening and fungus showing that ripening is less.  However, as the amount of incense burnt increases, the ripening speeds us as the softening and fungal infection show a marked increase.

Thus from the statistical table and t test, we have concluded that change in mass of fruit and the refractive index in % has no significant correlation between them.

The scientific justification for the above result is as follows:-

Ripening of Manilkara zapota is a process that is marked by several changes in the nature, firmness, texture, odour and content of the fruit.  A ripened fruit is softer, sweeter, different in colour and more aromatic compared to an unripened one.  Overripening can lead to senescence or dying of the fruit.   The presence of gases in Boswellia serrata hasten the ripening processs due to which it attains the same level of ripeness in a shorter duration of time.  When the time period is fixed, the fruits show varying amounts of ripeness.  Two factors which test ripeness are refractive index of fruit extract and decrease in mass of fruit.

• This experiment has a lesser impact of systematic error as the uncertainty of the measuring scale used is ±0.01.
• There are sufficient number of trials conducted to ensure that results are consistent and do not have large variations due to random error.
• The digital refractometer gives an accurate reading of refractive index reducing chances of human error.
• All significant factors which has a chance to affect the reliability of the data collected like  temperature, humidity and exposure time interval have been effectively controlled.

A possible extension to this experiment would be to conduct a further study on the effect of a greater number of incense sticks on ripening of sapodilla fruit. A similar experiment can also be considered for other fruit varieties like papaya, banana and mangoes.  The time period can be varied from 5 to 10 days to observe long term ripening effect The effect of refrigeration, salting and other preservation methods can also be studied.

For example, if we study effect of coldness on ripening, we can keep the refrigerator at different temperature settings (Max, Med, Min) to study its effect on fruit ripening.

Contento, G., et al., editors. Quality Control and Radiation Protection of the Patient in Diagnostic Radiology and Nuclear Medicine: Proceedings of a Workshop Held in Grado, Italy, September 29 to October 1 1993. Nuclear Technology Publ, 1995.

India, Nuffoods Spectrum. Natural Vs Artificial Fruit Ripening. https://nuffoodsspectrum.in/opinion/31/6928/natural-vs-artificial-fruit-ripening.html Accessed 30 Jan. 2021.

Iqbal, Noushina, et al. “Ethylene Role in Plant Growth, Development and Senescence: Interaction with Other Phytohormones.” Frontiers in Plant Science, vol. 8, Apr. 2017. PubMed Central, doi:10.3389/fpls.2017.00475.--. “Ethylene Role in Plant Growth, Development and Senescence: Interaction with Other Phytohormones.” Frontiers in Plant Science, vol. 8, Apr. 2017. PubMed Central, doi:10.3389/fpls.2017.00475.

Marrero, A., et al. “CONTROL OF ARTIFICIAL RIPENING OF BANANAS THROUGH ATMOSPHERE MODIFICATION AND REFRIGERATION.” Acta Horticulturae, no. 600, Mar. 2003, pp. 393–99. DOI.org (Crossref), doi:10.17660/ActaHortic.2003.600.57.

Nyanjage, M. O., et al. “A Comparative Study On the Ripening and Mineral Content of Organically and Conventionally Grown Cavendish Bananas.” Biological Agriculture & Horticulture, vol. 18, no. 3, Jan. 2001, pp. 221–34. DOI.org (Crossref), doi:10.1080/01448765.2001.9754885.

Prevention of Post-Harvest Food Losses Fruits, Vegetables and Root Crops a Training Manual - Pre-Harvest Factors in Produce Marketing-Perishability and Produce Losses-Cont. https://www.fao.org/3/T0073E/T0073E02.htm. Accessed 30 Jan. 2021.

Vaviya, Harshad, et al. “Identification of Artificially Ripened Fruits Using Machine Learning.” SSRN Electronic Journal, 2019. DOI.org (Crossref), doi:10.2139/ssrn.3368903.