ISOLATION AND EVALUATION OF SAPONIN EXTRACT FROM SOYMEAL AS A 
NATURALLY OCCURRING COUPLING AGENT FOR FIBER-BASED BIOCOMPOSITES 
FOR FOOD PACKAGING 

By 

Aidan McArdle 

A THESIS 

Submitted to 
Michigan State University 
in partial fulfillment of the requirements 
for the degree of 

Packaging – Master of Science 

2024 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ABSTRACT 

Soymeal is the prominent byproduct of soybean oil extraction and is mainly used as a 

high-protein source in animal feed. Saponins are also components of this soymeal. They have 

been associated with various health issues for livestock and are noted to negatively impact the 

taste and appeal to eat. The safe removal of saponins from soymeal can provide a sustainable 

source for saponins, and simultaneously provide a more desirable meal for livestock. The aim of 

this study was to investigate the feasibility of a saponin extract from soymeal as a naturally 

occurring coupling agent to improve the properties of fiber-based composites for food 

packaging. The first part of this study focused on determining the invasiveness of the saponin 

extraction on the meal. The isolation of saponins was proven via HPLC-MS, and a proximate 

composition analysis of soymeal post-extraction confirmed a nutritional content that was 

consistent with the initial meal. Second, this study focused on investigating the effectiveness of 

the saponin extract as a coupling agent. This effectiveness was shown for polylactic acid-soyhull 

composites based on an improvement of mechanical properties. The treatment of the filler with 

sulfuric acid prior to its inclusion into the polymer matrix improved the water vapor 

permeability, UV barrier, and thermal stability of the composites. With both treatments (saponin 

extract and acidification), mechanical, barrier, and thermal properties of composites were 

improved. This study shows that the saponin extract from soymeal has promise for potential as a 

coupling agent. 

 
 
 
 
 
 
 
 
 
 
This thesis is dedicated to my parents, brother, and grandparents. 

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ACKNOWLEDGEMENTS 

First, I would like to acknowledge Dr. Eva Almenar (School of Packaging, MSU) for her 

tireless support and guidance through all aspects of this thesis. Without her assistance and 

counsel, I do not think the accomplishment of this would have been possible. Beyond her support 

in the project, her experience, advice, and company have given me an invaluable and enjoyable 

experience for the past 2 years I would not trade. I would also like to thank my committee 

members in Dr. Laurent Matuana and Dr. Jeffrey Swada for their time and valuable insight into 

various aspects of the project and beyond. 

I would like to especially mention Haile Duguma for his never-ending guidance, 

availability, and company throughout this experience as well, he was an extremely valuable asset 

for every aspect of this thesis and an amiable friend through its completion. 

I am also grateful for the expertise and knowledge provided by Dr. Eric Gurzell (School 

of Food Science, MSU), he was instrumental in attaining and fully understanding many aspects 

of the project and went beyond necessity on multiple occasions to help.  

I am thankful to Guanqi Lu for performing statistical analyses for the data. 

I would like to acknowledge Aaron Walworth for his exceptional knowledge of the 

equipment, always offering help, and ensuring safety in the lab, he keeps the department afloat. 

We graciously thank the Michigan Soybean Committee for their financial support during 

this endeavor, a detailed tour of their oil extraction facility, and supplying the raw materials.  

I am grateful to my friends and acquaintances in the School of Packaging, including: 

Hazem, Saleh, Mohamed, Rishabh, Abi, Jim, Jayesh, Anurag, Nick, Robby, Muhammad, 

Aparajith, and Ben for their support, availability, and friendship along the way. I would like to 

give a shoutout to the dawgs, you know who you are. I would also like to express my gratitude 

for my other group members in Korey Fennel, and Purva Khule.  

Finally, I am eternally grateful for the endless support, guidance, and encouragement 

provided by my parents, brother, and family throughout the entirety of my schooling. Without 

them, none of this would have been possible, and I am beyond lucky to have them.

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TABLE OF CONTENTS 

CHAPTER 1 ….............................................................................................................................. 1 
INTRODUCTION …..................................................................................................................... 1 
1.1 Introduction ….............................................................................................................. 1 
1.2 Objectives …………………………............................................................................ 4 
1.3 Hypotheses ................................................................................................................... 5 
REFERENCES .............................................................................................................................. 6 

CHAPTER 2 …............................................................................................................................ 11 
LITERATURE REVIEW ............................................................................................................ 11 
2.1 Introduction to PLA ................................................................................................... 11 
2.2 Plastic Additives ........................................................................................................ 11 
2.2.1 Plasticizers .................................................................................................. 11 
2.2.2 Stabilizers .................................................................................................... 12 
2.2.3 Plastic Fillers ............................................................................................... 12 
2.2.3.1 Organic Fillers ............................................................................. 12 
2.2.3.1.1 Organic Fillers from Agricultural Waste ...................... 12 
2.2.3.1.2 Properties of Composites from Agricultural Waste ...... 13 
2.2.3.1.3 Applications .................................................................. 13 
2.2.3.2 Inorganic Fillers ........................................................................... 13 
2.2.3.3 Pre-Treatment of Fillers ............................................................... 14 
2.2.3.3.1 Surface Treatment of Organic Fillers ........................... 14 
2.2.3.3.2 Surface Treatments of Inorganic Fillers ....................... 15 
2.2.4 Coupling Agents ......................................................................................... 15 
2.2.4.1 Chemical (Commercial) Coupling Agents ................................... 16 
2.2.4.2 Naturally Derived Coupling Agents ............................................ 16 
2.3 Soy & Soybean Meal ................................................................................................. 17 
2.3.1 Soyasaponins ............................................................................................... 17 
2.3.2 Soy Isoflavones ........................................................................................... 18 
2.3.3 Soymeal as a Food Source .......................................................................... 18 
2.3.4 Nutritional Effect of Saponins & Isoflavones in Soymeal .......................... 18 
2.3.4.1 Soyasaponin & Isoflavone Effect on Flavor of Meal .................. 18 
2.3.4.2 Soyasaponin & Isoflavone Effect on Health of Livestock ........... 19 
2.3.4.2.1 Soyasaponin & Isoflavone Effect on Cattle .................. 19 
2.3.4.2.2 Soyasaponin & Isoflavone Effect on Fish .................... 19 
2.3.4.2.3 Soyasaponin & Isoflavone Effect on Swine ................. 20 
2.3.4.2.4 Soyasaponin & Isoflavone Effect on Poultry ................ 20 

2.3.5 Consequences of Removal of Soyasaponins & Isoflavones from 

Soymeal.....................................................................................21 
REFERENCES ............................................................................................................................ 22 

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CHAPTER 3 ................................................................................................................................ 32 
MATERIALS & METHODS ...................................................................................................... 32 
3.1 Extraction and Quantification of Saponin Derivative from Soymeal ........................ 32 
3.1.1 Materials ..................................................................................................... 32 
3.1.2 Extraction of Soyasaponin Derivative ........................................................ 32 
3.1.3 Quantification of Saponin in Derivation ..................................................... 33 
3.2 Renourishment of Soymeal ........................................................................................ 33 
3.2.1 Verification of Nutrient Consistency after Reintroduction ......................... 34 
3.2.1.1 Protein Content ............................................................................ 34 
3.2.1.2 Mineral/Ash Content .................................................................... 34 
3.2.1.3 Lipid Content ............................................................................... 34 
3.2.1.4 Moisture Analysis ........................................................................ 35 
3.2.1.5 Carbohydrate Content .................................................................. 35 
3.3 Preparation of Filler for Incorporation into Polymer Matrix ..................................... 35 
3.3.1 Materials ..................................................................................................... 35 
3.3.2 Particle Size Reduction of Soybean Hull Filler .......................................... 35 
3.3.3 Treatment of Powdered Soybean Hull Filler .............................................. 36 
3.4 Preparation/Fabrication of PLA/Soyhull Composites ............................................... 36 
3.5 Characterization of Composites ................................................................................. 37 
3.5.1 Mechanical Properties ................................................................................. 37 
3.5.2 Barrier Properties ........................................................................................ 38 
3.5.2.1 Oxygen Permeability ................................................................... 38 
3.5.2.2 Water Vapor Permeability ........................................................... 39 
3.5.3 Thermal Properties ...................................................................................... 39 
3.5.3.1 Thermal Gravimetric Analysis ..................................................... 39 
3.5.3.2 Differential Scanning Calorimetry (DSC) ................................... 39 
3.5.4 Morphological Analysis .............................................................................. 40 
3.5.5 Optical Properties ........................................................................................ 40 
3.5.5.1 Color ............................................................................................ 40 
3.5.5.2 Light Transmittance ..................................................................... 40 
3.6 Statistical Analysis ..................................................................................................... 40 
REFERENCES ............................................................................................................................ 41 

CHAPTER 4 ................................................................................................................................ 42 
RESULTS & DISCUSSION ....................................................................................................... 42 
4.1 Quantification of Soyasaponin Extract ...................................................................... 42 
4.2 Soymeal Nutrient Evaluation ..................................................................................... 42 

4.2.1 Proximate Composition Analysis of Soymeal After Extraction & 

Reintroduction .................................................................................................................. 42 
4.2.1.1 Protein Content ............................................................................ 43 
4.2.1.2 Mineral/Ash Content .................................................................... 43 

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4.2.1.3 Lipid Content ............................................................................... 43 
4.2.1.4 Moisture Content ......................................................................... 43 
4.2.1.5 Carbohydrate Content ………………………………………….. 44 
4.2.2 Saponin Content .......................................................................................... 44 
4.3 Characterization of PLA-Soybean Hull Composites ................................................. 45 
4.3.1 Mechanical Properties ................................................................................. 45 
4.3.2 Barrier Properties ........................................................................................ 47 
4.3.2.1 Oxygen Permeability ................................................................... 48 
4.3.2.2 Water Vapor Permeability ........................................................... 49 
4.3.3 Thermal Properties ...................................................................................... 50 
4.3.3.1 Thermogravimetric Analysis ....................................................... 50 
4.3.3.2 Differential Scanning Calorimetry (DSC) ................................... 51 
4.3.4 Morphological Analysis .............................................................................. 53 
4.3.5 Optical Characterization ............................................................................. 54 
4.3.5.1 Color ............................................................................................ 54 
4.3.5.2 Light Transmittance ..................................................................... 55 
REFERENCES ............................................................................................................................ 56 

CHAPTER 5 ................................................................................................................................ 59 
CONCLUSION ............................................................................................................................ 59 
5.1 Conclusion ................................................................................................................. 59 
5.2 Future Work ............................................................................................................... 60 

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CHAPTER 1 

INTRODUCTION 

1.1 Introduction  

The need and use for plastics in food packaging has increased over 400% over the last 50 

years [1]. The primary plastics used in this food packaging take upwards of 400 years to degrade 

biologically [2]. This poor biodegradability is cause for ecological and humanitarian concern, 

ranging from toxic garbage patches covering the oceans to microplastics contaminating humans’ 

bloodstream [3]. 

Although a reduction or overall elimination of plastic use seems like a simple solution, 

plastics in food packaging offer light weights, desirable aesthetics, cheap and low processability, 

and provide more than capable strength and barrier properties for protection and extension of 

food shelf life [4 - 6]. Beyond this, the high energy requirements for alternative or reusable 

packaging formats often outweigh the environmental impact they may be thought to provide in 

the long term [7, 8].  Eliminating plastics entirely from food packaging is not currently a feasible 

solution to the problem and therefore, the investigation of adjusting them, or substituting for 

more eco-friendly alternatives has become paramount. 

One of the most promising materials for the future of food packaging has been polylactic 

acid (PLA). PLA is a biodegradable, industrially compostable plastic derived from polymerized 

fermented lactic acid from crops including corn, potatoes, and sugar. It shows potential for the 

future of packaging materials due to its abundant and sustainable source, good strength, and ease 

of processibility. However, it typically shows low thermal stability, minimal toughness, and 

modest moisture barrier properties [9]. While it can be industrially composted in days, neat PLA 

can still take upwards of 80 years to decompose in nature [10].  

Fillers are materials that are inserted into a polymer matrix as a direct partial substitute 

for raw plastic [11]. They are often incorporated to reduce raw plastic use and thus cost of 

manufacturing, while ideally retaining performance comparative to the neat polymer. Some 

fillers have even been found to induce antimicrobial [12, 13], antioxidant [13], and antifungal 

[14] properties, improving the capacity to prevent product spoilage as a food packaging material.  

1 

 
 
 
 
 
 Agricultural waste is defined as the unwanted or unusable materials produced from 

agricultural operations immediately relating to the growing of crops or the raising of animals 

[15].  Agricultural waste has been a popular filler source due to its low cost and chemical 

composition, making it more desirable for industrially produced composites [16]. Agriculturally 

sourced fillers have been consistently found to improve biodegradation rate [17 - 22] and have 

occasionally resulted in similar or improved barrier properties to water [23] and oxygen [24]. 

The drawback in using agricultural waste-based fillers has consistently been a reduction in 

tensile properties, mainly toughness and elongation [25, 26]. Typically, these decreases in 

mechanical properties are attributed to the reactive groups on the filler surface not interacting 

with the polymer main chain, causing gaps and overall disruption in the uniformity and 

compatibility of the matrix, thus decreasing the overall strength [27, 28].  

Treatments to both the waste and the polymer have been investigated in attempt to 

combat these deteriorative properties. The most common treatments include the acidification or 

alkalinization of the waste, and/or the incorporation of a coupling agent [29]. Acidification and 

alkalinization treatments involve exposing the filler to highly acidic or basic environments, 

respectively, with the idea of removing impurities, changing the morphological structure of the 

particles, and/or otherwise promoting better adhesion into a polymer chain [30]. Various studies 

have concluded in the improvement in properties of organic waste composites with PLA as a 

result of these filler treatments [30, 31]. 

Coupling agents are compounds that form chemical bonds between dissimilar molecules, 

working to provide better adhesion and interfacial bonding between a polymer matrix and non-

polymer components [32]. The typical structural requirements for coupling agents involve 

having two types of functional groups, one able to copolymerize with the desired polymer, and 

one able to strongly adhere to the foreign material used [33].  

The extent and potential for coupling agents in the future of packaging is noteworthy, 

particularly in the future of filler-based plastics. Perhaps the main restriction in the use of 

coupling agents in this field is the increased cost to incorporate, their chemical origin, and the 

effect they can have on the biodegradability of the polymer. In the area of filler-based 

composites, common commercially available coupling agents such as methylene diphenyl 

diisocyanate (MDI), toluene diisocyanate (TDI), silane, and maleic anhydride have been known 

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to successfully improve mechanical properties but end up notably decreasing the 

biodegradability [34 - 36]. There have been investigations of naturally derived coupling agents 

with moderate mechanical improvements and non-disruption of biodegradability, including pine 

rosin, palm and sesame oils, and fatty acids including hexanoic, octanoic, decanoic, and 

dodecanoic acids [37, 38]. However, these are extracted invasively and unsustainably, show 

minute improvements, and have little potential to be applied on a higher production scale.  

Saponins are triterpene glycosides naturally found in many plants, having a particularly 

noteworthy presence in soapwort, licorice, and soy [39, 40]. Saponins are long molecules, 

characterized by both largely hydrophilic and hydrophobic ends, giving them excellent surfactant 

and emulsification capabilities [41]. Isolated saponins have been heavily investigated in 

pharmaceutical and fields, and have been cited to contain anti-inflammatory, anti-cancer, 

antimicrobial, antioxidant, and cholesterol-inhibiting capabilities [42-46]. Based on the structural 

requirements above for viable coupling agents, saponins can be considered with great potential 

for this application as well. Saponins from soap berry have been investigated for potential as a 

coupling agent in the use of polypropylene composites incorporated with nanocellulose with 

some success [47]. Although all varieties of saponin differ based on their genus of origin, 

saponins derived from soy and soy related products, deemed “soyasaponins”, are believed to 

have equal promise for these types of applications.  

Soybeans are one of the most grown crops worldwide, with global production reaching 

more than 370 million tons in 2021 [48]. It is estimated that more than 85% of this yield is used 

in the extraction of soybean oil, which is widely used for biofuel and cooking oil [49]. During 

this extraction process, the raw beans are subject to repeated exposure to heat and hexane 

immersion, removing lipids and leaving behind byproducts such as “defatted soymeal”, and 

“soyhulls”. In the United States, 97% of soymeal is sold directly to farmers or feed supply stores 

for use as a high protein animal feed for cattle, poultry, fish, and swine, while the remaining 3% 

is used for derived protein concentrate or soymilk [50]. Soyhulls are typically either added to the 

meal for their fibrous content or are disposed of. The increase in soybean oil extraction facilities 

has resulted in a large influx of both these byproducts, requiring additional uses for them [51]. 

Soymeal is considered a nutritious and growth inducing formula for these livestock due 

to its high digestible protein and carbohydrate content, promoting health, energy, growth, and 

3 
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productivity. Although it is widely ingrained into farm animal’s diets, there have been numerous 

findings that some aspects of it may induce adverse health effects. For instance, saponins and 

isoflavones in the meal have been known to cause ruptures in intestinal tracts, decreased nutrient 

digestion and appetite, and decreased embryonic health and development in bovine mammals 

and swine [52 - 56]. Saponins in feed have also been heavily attributed to inducing fatal enteritis 

and severely reduced nutrient digestion in fish. [57, 58]. Furthermore, the bitter flavors these 

compounds induce have been shown to reduce many of the animal’s desire to consume the meal 

at all [59 - 61]. Based on these findings, a soymeal void of these compounds would provide all 

the positive aspects regarding its nutritional proficiency as a food source, without the negative 

health effects often involved. A safe and minimally invasive extraction of these compounds 

would result in a saponin isolate removed from the meal, and an overall healthier food product. It 

would also provide merit for a bountiful, sustainable, and easily accessible source for these 

compounds for use beyond packaging. 

This thesis aims to establish a sustainable and naturally sourced coupling agent with a 

minimally invasive procedure that can even improve the raw material it was extracted from. It 

investigates the effectiveness of the proposed compound on soyhull-PLA composites. 

Additionally, it assesses the effect of acidification of the filler on the performance of the 

proposed coupling agent. This study provides a baseline for improving the properties of green 

plastic composites without disrupting the “green” aspect of them. 

1.2 Objectives 

The purpose of this study is first, to demonstrate that saponins can be safely extracted 

from defatted soymeal without damaging the physical or nutritional integrity of it, allowing for 

its continuation to be sold as an improved, high-protein food source for animals. 

The second aim of this study is to determine the efficacy of saponin extract to be used as 

a naturally occurring coupling agent for PLA and an agricultural waste filler of crushed soybean 

hulls. 

Finally, it is to determine the effect of acid-treatment of the soyhull filler on the resultant 

composite properties and the performance of the saponin coupling agent. 

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1.3 Hypotheses 

1.  Saponins can be safely extracted from defatted soymeal without damaging or otherwise 

interrupting the physical and nutritional integrity of the meal, leaving it fit for 

consumption. 

2.  Saponins can function as a naturally occurring coupling agent for a PLA-soybean hull 

composite to improve the overall mechanical properties of the composite. 

3.  The use of sulfuric acid (H2SO4) during treatment of the crushed soybean hull filler with 

the saponin coupling agent will improve the extent to which the saponins interact with the 

filler particles. 

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REFERENCES 

Facts and figures about materials, waste and recycling, Containers and Packaging, 

1. 
Product Specific Data. United States EPA, (2022). 

Hughes, A.M., How long it takes everyday items to decompose, The Waste Management 

2. 
Recycling Coalition. (2022). 

Leslie, H.A., Van Velzen, M.J.M., Brandsma, S.H., Vethaak, A.D., Garcia-Vallejo, J.J., 

3. 
Lamoree, M.H., Discovery and quantification of plastic particle pollution in human blood, 
Environment International. 163 (2022) 107199. doi:10.1016/j.envint.2022.107199.  

Evans, D.M., Parsons, R., Jackson, P., Greenwood, S., Ryan, A., Understanding plastic 

4. 
packaging: The co-evolution of materials and Society, Global Environmental Change. 65 (2020) 
102166. doi:10.1016/j.gloenvcha.2020.102166.  

Coles, R., Kirwan, M.J., McDowell, D., Food Packaging Technology, Blackwell, Oxford, 

5. 
2003.  

Kirwan, M.J., Food and Beverage Packaging Technology, Wiley-Blackwell, Chichester, 

6. 
West Sussex, 2011. DOI:10.1002/9781444392180  

Khoo, H.H., Tan, R.B., Environmental impacts of conventional plastic and bio-based 

7. 
carrier bags, The International Journal of Life Cycle Assessment. 15 (2010) pp 338–345. 
doi:10.1007/s11367-010-0163-8.  

Accorsi, R., Battarra, I., Guidani, B., Manzini, R., Ronzoni, M., Volpe, L., Augmented 

8. 
spatial LCA for comparing reusable and recyclable food packaging containers networks, Journal 
of Cleaner Production. pp 375 (2022) 134027. doi:10.1016/j.jclepro.2022.134027 

Singha, S., Hedenqvist, S.H., A review on barrier properties of poly(lactic acid)/clay 

9. 
nanocomposites, Polymers. 12 (2020) 1095. doi:10.3390/polym12051095.  

10. 

11. 

PLA Decomposition Rate, Gianeco Plastics. (2013)  

Bio-based fillers provide eco-friendly solutions, AZoMaterials. (2021).  

12. 
Kumar, T.S., Chandrasekar, M., Senthilkumar, K., Ilyas, R.A., Sapuan, S.M., Hariram, 
N., Varada Rjulu, A., Siengchin, S., Characterization, thermal and antimicrobial properties of 
hybrid cellulose nanocomposite films with in-situ generated copper nanoparticles in Tamarindus 
indica nut powder, Journal of Polymers and the Environment. 29 (2020) pp 1134–1142. 
doi:10.1007/s10924-020-01939-w.  

S. Roy, J.-W. Rhim, Antioxidant and antimicrobial poly(vinyl alcohol)-based films 

13. 
incorporated with grapefruit seed extract and curcumin, Journal of Environmental Chemical 
Engineering. 9 (2021) 104694. doi:10.1016/j.jece.2020.104694.  

14. 
F.V. Ferreira, M. Mariano, L.S.S. Lepesqueur, I.F. Pinheiro, L.G. Santos, J. Burga-
Sánchez, Souza, D.H.S., Koga-Ito, C.Y., Teixiera-Neto, A.A., Mei, L.H.I., Gouveia, R.F., Lona, 
L.M.F., Silver nanoparticles coated with dodecanethiol used as fillers in non-cytotoxic and 

6 
6 

 
 
 
 
 
 
antifungal PBAT surface based on nanocomposites, Materials Science and Engineering: C. 98 
(2019) pp 800–807. doi:10.1016/j.msec.2019.01.044.  

15. 

Brenner, K., Agricultural Waste, South Coast Air Quality Management District, (2017)   

Akinlabi, O., Umunakwer, R., Madueke, C.I.., A Review on Agricultural Wastes as Filler 

16. 
for Vulcanized Natural Rubber, Journal of Institute of Polymer Engineers (JIPE) Vol. 1 (2017) 
pp 23-34   

17. 
Huang, Z., Qian, L., Yin, Q., Yu, N., Liu, T., Tian, D., Biodegradability studies of 
Poly(Butylene Succinate) composites filler with sugarcane rind fiber, Polymer Testing, Vol. 66 
pp 319-326 (April, 2018). https://doi.org/10.1016/j.polymertesting.2018.02.003.  

18.  Meereboer, K.W., Pal, A.K., Cisneros-López, E.O., Misra, M., Mohanty, A.K., The effect 
of natural fillers on the marine biodegradation behaviour of poly(3-hydroxybutyrate-co-3-
hydroxyvalerate) (PHBV). Sci Rep 11, 911 (2021). https://doi.org/10.1038/s41598-020-78122-7  

Bayerl, T., Geith, M., Arcut, A., Bhattacharyya, D., Influence of fibre architecture on the 

19. 
biodegradability of FLAX/PLA composites, International Biodeterioration & 
Biodegradation,Volume 96, (2014), pp 18-25, ISSN 0964-8305, 
https://doi.org/10.1016/j.ibiod.2014.08.005   

Kumar, R., Yakubu, M.K., Anandijiwala, R.D., “Biodegradation of flaxfiber reinforced 

20. 
Polylactic Acid”, Express Polymer Letter Vol. 4 No. 7, pp 423-430 (2010), DOI: 
10.3144/expresspolymlett.2010.53    

Lee, S.H., Wang, S., Biodegradable polymers/bamboo fiber biocomposite with bio-based 

21. 
coupling agent, Composites Part A: Applied Science and Manufacturing. 37 pp 80–91. (2006) 
doi: 10.1016/j.compositesa.2005.04.015.   

Varyan, I., Kolesnikova, N., Xu, H., Tyubaeva, P., Popov, A., Biodegradability of 

22. 
polyolefin-based compositions: Effect of natural rubber, Polymers. 14 (2022) 530. 
doi:10.3390/polym14030530.  

Travalini, A.P., Lamsal, B., Magalhães, W.L., Demiate, I.M., Cassava starch films 

23. 
reinforced with lignocellulose nanofibers from cassava bagasse, International Journal of 
Biological Macromolecules. 139 (2019) pp 1151–1161. doi:10.1016/j.ijbiomac.2019.08.115.  

24. 
Terzioğlu, P., Güney, F., Parın, F.N., Şen, I., Tuna, S., Biowaste Orange Peel 
Incorporated Chitosan/Polyvinyl Alcohol Composite Films for Food Packaging Applications, 
Food Packaging and Shelf Life. 30 (2021) 100742. doi:10.1016/j.fpsl.2021.100742.  

25. 
Álvarez-Chávez, C.R., Sánchez-Acosta, D.L., Encinas-Encinas, J.C., Esquer, J., 
Quintana-Owen, P., Madera-Santana, T.J., "Characterization of Extruded Poly(lactic acid)/Pecan 
Nutshell Biocomposites", International Journal of Polymer Science, vol. 2017, Article ID 
3264098, (2017). https://doi.org/10.1155/2017/3264098  

Fehlberg, J., Lee, C.L., Matuana, L., Almenar, E., Orange peel waste from juicing as raw 

26. 
material for plastic composites intended for use in food packaging Journal of Applied Polymer 
Science, 137 (2020), p 48841, https://doi.org/10.1002/app.48841   

7 
7 

 
 
 
 
 
 
Techniczne, C., The effect of selected natural fillers on the mechanical properties of low-

27. 
density polyethylene, (2017). doi:10.4467/2353737xct.17.181.7289. 

Ramesh, M., Rajeshkumar, L.N., Srinivasan, N., Kumar, D.V., Balaji, D., Influence of 

28. 
filler material on properties of fiber-reinforced polymer composites: A Review, E-Polymers. 22 
(2022) pp 898–916. doi:10.1515/epoly-2022-0080.  

Duguma, H.T., Khule, P., McArdle, A., Fennell, K., Almenar, E., Turning agricultural 

29. 
waste into packages for food: A literature review from origin to end-of-life, Food Packaging and 
Shelf Life. 40 (2023) 101166. doi:10.1016/j.fpsl.2023.101166.  

Zhou, L., Ke, K., Yang, M.B., Yang, W., Recent progress on chemical modification of 

30. 
cellulose for high mechanical-performance poly(lactic acid)/cellulose composite: A Review. 
Composites Communications, 23, 100548 (2021). https://doi.org/10.1016/j.coco.2020.100548  

31. 
SÖĞÜT, E., SEYDİM, A.C., Utilization of kiwi peel lignocellulose as fillers in 
poly(lactic acid) films, Journal of the Turkish Chemical Society Section A: Chemistry. 9 (2022) 
pp 283–294. doi:10.18596/jotcsa.1024326.  

32.  Mangore, R.V., Ahankari, S.S., Verma, K.D., Kar, K.K., Fly ash-reinforced acrylonitrile 
butadiene styrene composites, Handbook of Fly Ash. (2022) pp 301–334. doi:10.1016/b978-0-
12-817686-3.00026-8.  

Coupling Agents: Strengthening the Weakest Link in the Composites Chain. ALTANA 

33. 
(2011).  

34. 
Zamir, S.S., Fathi, B., Ajji, A., Robert, M., Elkoun, S., Biodegradation of modified 
starch/poly lactic acid nanocomposite in soil Polymer Degradation Stability, 199, Article 109902 
(2022) 10.1016/j.polymdegradstab.2022.109902  

Roumeli, E., Terzopoulou, Z., Pavlidou, E., Chrissafis, K., Papadopoulou, E., 

35. 
Athanasiadou, E., Triantafyllidis, K., Bikiaris, D.N., Effect of maleic anhydride on the 
mechanical and thermal properties of hemp/high-density polyethylene green composites, Journal 
of Thermal Analysis and Calorimetry. 121 (2015) pp 93–105. doi:10.1007/s10973-015-4596-y.  

Rakmae, S., Ruksakulpiwat, Y., Sutapun, W., Suppakarn, N., Effect of silane coupling 

36. 
agent treated bovine bone based carbonated hydroxyapatite on in vitro degradation behavior and 
bioactivity of PLA Composites, Materials Science and Engineering: C. 32  pp 1428–1436. 
(2012) doi:10.1016/j.msec.2012.04.022.  

Inga-Lafebre, J., Pulido-González, H., González-Núñez, R., Hernández-Hernández, M.E., 

37. 
Rabelero-Velasco, M., Aranda-García, F.J., Jasso-Gastinel, C.F., The multirole of modified 
natural gums for multicomponent polymers: As coupling agents for polymers reinforced with 
cellulosic fibers or compatibilizers for biodegradable polymer blends, Química Nova. (2019). 
doi:10.21577/0100-4042.20170333.  

Chun, K., Husseinsyah, S., Osman, H., Preliminary Study of Palm Oil Based Coupling 

38. 
Agent for Polypropylene/Cocoa Pod Husk Composites. Adv. Environ. Biol., 8(8), pp 2640-2644, 
(2014) 

8 
8 

 
 
 
 
 
 
Fenwick, D.E., Oakenfull, D., Saponin content of food plants and some prepared foods, 

39. 
Journal of the Science of Food and Agriculture. 34 (1983) pp 186–191. 
doi:10.1002/jsfa.2740340212.  

Vincken, J.P., Heng, L., de Groot, A., Gruppen, H., Saponins, classification and 

40. 
occurrence in the plant kingdom, Phytochemistry. 68 (2007) pp 275–297. 
doi:10.1016/j.phytochem.2006.10.008.  

41.  Moses, T., Kalliope, K., Osbourn, A., Metabolic and functional diversity of saponins, 
biosynthetic intermediates and semi-synthetic derivatives, Critical Reviews in Biochemistry and 
Molecular Biology, 49:6, pp 439-462, (2014) doi:10.3109/10409238.2014.953628  

42. 
Spagnuolo, C., Russo, G.L., Orhan, I.E., Habtemariam, S., Daglia, M., Sureda, A., 
Nabavi, S.F., Devi, K.P., Loizzo, M.R., Tundis, R., Nabavi, S.M., Genistein and cancer: Current 
status, challenges, and Future Directions, Advances in Nutrition. 6 (2015) pp 408–419. 
doi:10.3945/an.114.008052.  

Omar A., Kalra R.S., Putri, J., Elwakeel, A., Kaul, A.C., Wadhwa, R., Soyasapogenol-A 

43. 
targets CARF and results in suppression of tumor growth and metastasis in p53 compromised 
cancer cells. Sci Rep 10, 6323 (2020). https://doi.org/10.1038/s41598-020-62953-5  

44. 
Chen, J., Ullah, H., Zheng, Z., Gu, X., Su, C., Xiao, L., Wu, X., Xiong, F., Li, Q., Zha, 
L., Soyasaponins reduce inflammation by downregulating MyD88 expression and suppressing 
the recruitments of TLR4 and MyD88 into lipid rafts. BMC Complement Med Ther 20, 167 
(2020). https://doi.org/10.1186/s12906-020-2864-2  

Guang, C., Chen, J., Sang, S., Cheng, S., Biological functionality of Soyasaponins and 

45. 
Soyasapogenols, Journal of Agricultural and Food Chemistry. 62 (2014) pp 8247–8255. 
doi:10.1021/jf503047a.  

46. 
Iwashita, Y., Suzuki, N., Matsunari, H., Sugita, T., Yamamoto, T., Influence of soya 
saponin, soya lectin, and cholyltaurine supplemented to a casein-based semipurified diet on 
intestinal morphology and biliary bile status in fingerling rainbow trout Oncorhynchus mykiss, 
Fisheries Science. 75 (2009) pp 1307–1315. doi:10.1007/s12562-009-0158-1.  

Cherian, B.M., Leão, A.L., Da Caldeira, M., Chiarelli, D., de Souza, S.F., Narine, S., 

47. 
Chaves, M.R.M., Use of Saponins as an effective surface modifier in cellulose nanocomposites, 
Molecular Crystals and Liquid Crystals. 556 (2012) pp 233–245. 
doi:10.1080/15421406.2012.635969.  

48.  Wunsch, N.G., Most grown crops in the world 2021, Statista. (2021).   

Cooking oil market - share, Trends & Industry Overview, Cooking Oil Market - Share, 

49. 
Trends & Industry Overview. (2023)   

USDA Coexistence Fact Sheet,  Uses for soybeans, United States Department of 

50. 
Agriculture (USDA). (Feb. 2015).   

Gerlt, S., (2023, January 3). Economist’s angle: Soybean processing growth is crushing 

51. 
it. Economist’s Angle: Soybean Processing Growth is Crushing It.  

9 
9 

 
 
 
 
 
 
Das, T., Banerjee, D., Chakraborty, D., Pakhira, M., Shrivastava, B., Kuhad, R., Saponin: 

52. 
Role in Animal System, Veterinary World. 5 (2012) 248. doi:10.5455/vetworld.2012.248-254.  

Grgic, D., Varga, E., Novak, B., Müller, A., Marko, D., Isoflavones in animals: 

53. 
Metabolism and effects in livestock and occurrence in feed, Toxins. 13 (2021) 836. 
doi:10.3390/toxins13120836.  

Shore, L.S., Rios, C., Marcus, S., Bernstein, M., Shemesh, M., Relationship between 

54. 
peripheral estrogen concentrations at insemination and subsequent fetal loss in cattle, 
Theriogenology. 50 (1998) pp 101–107. doi:10.1016/s0093-691x(98)00117-4.  

55. 
Smith, B.N., Dilger, B., Immunomodulatory potential of dietary soybean-derived 
isoflavones and saponins in pigs1, Journal of Animal Science. 96 (2018) pp 1288–1304. 
doi:10.1093/jas/sky036.  

Department of Animal Science - Plants poisonous to livestock, Cornell University 

56. 
Department of Animal Science. (2008).   

57. 
Iwashita, Y., Suzuki, N., Matsunari, H., Sugita, T., Yamamoto, T., Influence of soya 
saponin, soya lectin, and cholyltaurine supplemented to a casein-based semipurified diet on 
intestinal morphology and biliary bile status in fingerling rainbow trout Oncorhynchus mykiss, 
Fisheries Science. 75 (2009) pp 1307–1315. doi:10.1007/s12562-009-0158-1.  

58. 
Knudsen, D., Røn, Ø., Baardsen, G., Smedsgaard, J., Koppe, W., Frøkiær, H., 
Soyasaponins resist extrusion cooking and are not degraded during gut passage in Atlantic 
salmon (salmo salar L.), Journal of Agricultural and Food Chemistry. 54 (2006) pp 6428–6435. 
doi:10.1021/jf0604992.  

59.  Mikołajczyk-Bator, K., The significance of Saponins in shaping the quality of food 
products from Red Beet, Acta Scientiarum Polonorum Technologia Alimentaria. 21 (2022) pp 
81–90. doi:10.17306/j.afs.1012.  

60.  McLaughlin, L., Baile, A., Buckholtz, L., Freeman, K., Preferred flavors and 
performance of weanling pigs. Journal of Animal Science. (1983) 56: pp 1287-1293.  

Forbes, M., Voluntary Food Intake and Diet Selection in Farm Animals. Walingford: 

61. 
CABI. p 245 (1995). 

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CHAPTER 2 

LITERATURE REVIEW 

2.1 Introduction to PLA 

Poly lactic acid or PLA is a promising material currently at the forefront of alternative 

and sustainable polymers in place of traditional polyolefins [1, 2]. Low molecular weight PLA is 

synthesized by either chemical synthesis or bacterial fermentation of lactic acid from sugars of 

crops such as corn, cassava, sugarcane, sugarbeet, and maize [1, 2]. 

The polymerization of higher molecular weight PLA is seen to be done in one of three 

separate processes: direct condensation polymerization, direct polycondensation in an azeotropic 

medium, or lactide formation polymerization. The latter is by far the most seen on the industrial 

scale. Lactide formation is achieved through the removal of water under mild conditions without 

solvent. Generally, L-lactide, a product of the L-lactic acid, is used in the mass production of 

PLA, thus the alternative names Poly (L-lactide) or Poly Lactide. [1]. PLA can be produced in a 

wide array of structures, ranging from fully amorphous to highly crystalline.  

PLA is observed as a highly prominent plastic for the future of plastic manufacturing due 

to its sustainable and widely available source, ease of processing, and effective biodegradation. 

However, it has multiple characteristic contingencies restricting its potential for use, including 

low flexibility, thermal resistance, and gas barrier properties [3, 4]. 

2.2 Plastic Additives 

With the intention of improving performance, lowering the cost of production, reducing 

energy use, or promoting higher biodegradability, the use of additives is often employed to 

polymer matrices. These include additives such as plasticizers, fillers, compatibilizers, and 

stabilizers [5].  

2.2.1 Plasticizers 

Plasticizers are often incorporated into polymer matrices for improved flexibility and 

malleability, traits that are sought after in the production of plastic materials. These are typically 

low molecular weight compounds that are compatible with the plastic resin of use [6]. 

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2.2.2 Stabilizers 

Stabilizers are compounds introduced into polymer matrices with the desired intent of 

increasing the plastic’s resistance to certain degradation mechanisms both during processing and 

use. These envelop a variety of functional stabilizers, including heat stabilizers [7], light 

stabilizers [8], and oxidation stabilizers [9]. These additives come in a variety of forms and are 

most often employed in mechanical or civil engineering applications where materials may be 

subject to excessive conditions.  

2.2.3 Plastic Fillers 

Fillers are additive compounds typically in the micro or nano size that are incorporated 

with a polymer as a means to promote a large variety of performance enhancements, or act as a 

substitute for raw plastic to reduce cost [10]. Fillers can come in a near infinite range of forms 

and sources and can be responsible for improving properties such as thermal and UV light 

resistance, biodegradation, antimicrobial capability, antioxidant capacity, barrier properties, and 

mechanical properties, depending on the type of filler used [10].  Based on their nature of origin, 

fillers can generally be classified into two categories: organic and inorganic. 

2.2.3.1 Organic Fillers 

Organic fillers are particles or fibers incorporated into polymer matrices that have been 

derived from living, or what was once living matter [11]. Most organic fillers seen in recent 

literature have been derived from plant material, including wood and other fibrous green matter 

[12], and occasionally from animal material, including shells from eggs or even crustaceans [13]. 

2.2.3.1.1 Organic Fillers from Agricultural Waste 

Byproducts from agricultural materials and processes, deemed “agricultural waste”, are 

the main source of organic fillers [10]. Examples of waste used as organic fillers include corn 

stalk [14], fruit peels [15] [16], rice husks [17], coffee grounds [18], wheat straw [19], nutshells 

[17], and eggshells from chickens [13]. The reasons these are common sources for fillers are 

abundance, and exceptionally low costs. A low-cost partial replacement in plastic without the 

degradation of properties can be an important economic incentive for the eventual adaptation of 

this technology into industry. 

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2.2.3.1.2 Properties of Composites from Agricultural Waste 

Plastics incorporated with fillers derived from agricultural waste have consistently 

resulted in more brittle properties in comparison to neat plastics. This is resembled by 

improvements in stiffness, with reductions in percent elongation, flexural strength, and tensile 

strength [10, 11]. These fillers have also been acknowledged to reduce the barrier properties of 

the resultant composites to both water [14, 17], and to a lesser extent, oxygen [17, 20]. Fillers 

from agricultural sources have been attributed to decreases in thermal stability as well [17, 21]. 

These property reductions are generally attributed to the hydrophilic surface nature of a majority 

of these fillers, resulting in poor interfacial adhesion, and gaps in the hydrophobic polymer 

matrix [22]. While the poor inclusion into the polymer matrix for these fillers results in poorer 

mechanical and barrier properties, it often provides easier access for biodegradation mechanisms 

to decompose the material at end of life, resulting in quicker biodegradation in natural settings 

[23, 24]. 

2.2.3.1.3 Applications 

The increases in stiffness and decreases in thermal stability for composites involving 

agriculturally sourced fillers leave them only considered for non-packaging and insignificant 

applications in automotive, aircraft, leisure, and electronic industries [25]. These fillers are 

typically able to be incorporated at higher loadings than others due to their softer and 

nonabrasive nature [25, 26]. This allows for a higher substitution rate, reducing the raw plastic 

needed for composite production. The earlier deteriorations, along with low barrier properties 

associated with fillers from organic nature leaves very little application in food packaging 

formats for them. The majority of the investigation for its potential as a food packaging material 

has remained in academia. 

2.2.3.2 Inorganic Fillers 

Inorganic fillers are particles formed or derived from materials non-associated with living 

matter. The majority of these fillers are derived from minerals found in nature and finely ground. 

the most common inorganic fillers have been calcium carbonate (CaCO3) [27, 28], talc powders 

[29], and nano clays [30].  Inorganic fillers have been associated with inducing a number of 

effects on plastic properties, and occasionally providing new ones. Similar to organic fillers, the 

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use of inorganic fillers also typically induces greater stiffness, along with reductions in percent 

elongation, and flexural and tensile strength [28, 30-32]. This is also largely attributed to the 

surface chemistry of the fillers being incompatible with that of the polymer. Some inorganic 

fillers have however been cited to improve the thermal resistance of the resultant composite [31-

32]. There are cases where inorganic fillers have been observed to have minimal effect, or even 

improve the barrier properties of films as well [31-32] [35]. Some less common, or chemically 

produced inorganic fillers have been cited to induce plastics equipped with antimicrobial activity 

[33-34]. Inorganic fillers have also been observed to improve the biodegradation rate of the 

composite [35-36]. Due to the improved properties associated with inorganic fillers in relation to 

organic fillers, these types of additives are seen more often in industrial and packaging settings. 

The abundance in nature, and relatively cheap cost results in a similar cost-based incentive for 

mass production and incorporation that could be seen in organic filler polymers [27, 37]. 

2.2.3.3 Pre-Treatment of Fillers 

Due to the excessive deteriorative properties of polymers when incorporated with fillers 

of both organic and inorganic nature, surface treatments to the filler are often applied to promote 

a better  relationship between them and the plastic [38]. The most common treatment for reducing 

the hydrophilicity of all filler particles is the removal of as much water from them as possible. 

These drying mechanisms are typically done via hot drying [17, 20, 39], lyophilizing [40, 41], or 

spray drying [40, 42]. All three have been noted to improve dispersity and porosity in 

comparison to non-dried fillers [40, 41]. Beyond the removal of water, various chemical 

treatments work to alter the filler particles themselves.  

2.2.3.3.1 Surface Treatment of Organic Fillers 

There are a variety of chemical treatments for organic fillers prior to their inclusion into a 

polymer matrix as a mechanism of improving their compatibility with the plastic. 

Alkalinization is employed to reduce organic matter into more basic components, it is a 

common technique for the isolation of cellulose from other constituents such as lignin and 

hemicellulose in many agricultural sourced fillers [43-45]. The most common chemicals used in 

the alkalinization of filler particles are NaClO, [46] and NaOH [47]. Alkalinization has been 

14 
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proven to improve the acceptance of filler particles into various polymer matrices, resulting in 

higher tensile and barrier properties [45, 48].  

Acidification is also often employed to isolate or deteriorate cellulose structures from 

other contents in filler materials depending on the acid used [49]. The most common acids used 

in the pretreatment acidification process for fillers include HCl [50], H2SO4 [50], oxalic acid 

[51], formic acid [52], or a combination of them [51, 52]. Acidification has also been seen to 

improve the mechanical properties of organic filler-based composites, often increasing tensile 

strength and stiffness of resultant composites [53, 54]. 

Regardless of which treatment is used, the isolation or degradation of cellulose from the 

other components of many organic fillers allows for a more hydrophobic surface chemistry. The 

improvements in tensile properties associated with these treated fillers is largely attributed to 

this, cited to improve dispersion and compatibility with the polymer. 

2.2.3.3.2 Surface Treatments of Inorganic Fillers 

The most common surface modifiers for fillers such as CaCO3, talc, and nano clays 

involve the replacement of the hydrophilic reactive groups with hydrophobic ones. This can be 

achieved in a number of ways or a combination of them. In industry and academia, the most 

common methods for this involve high intensity thermal treatments such as gamma irradiation 

[55], stearic acid coatings, [56-58] and phosphate ester treatments [59].  

The treatment with stearic acid phosphate esters, and irradiation, have all been proven to 

show improvements in tensile strength and % elongation [55, 57-59]. These improvements are 

attributed to the altering of surface chemistry, resulting in better dispersion and compatibility of 

fillers within the matrix [58, 59]. 

2.2.4 Coupling Agents 

Coupling agents envelop a wide range of compounds that work to bind dissimilar 

materials together in unconventional environments [38]. They have been incorporated in polymer 

science in the form of combining multilayer plastic films and sheets, as well as in the inclusion 

of other additives [60]. They work to provide a strong linkage between the polar and nonpolar 

substituents and thus vastly improve mechanical and barrier properties. They have been widely 

15 
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used in filler-based composite resins for both organic and inorganic fillers as a means of 

improving resultant mechanical properties. 

2.2.4.1 Chemical (Commercial) Coupling Agents 

Some of the most common coupling agents used in polymer-additive relationships are 

silane [61], maleic anhydride [62], methylene diphenyl diisocyanate (MDI) [63] or derivatives of 

these [64].  Some coupling agents, such as maleic anhydride are grafted onto the polymer before 

filler loading [65], others such as MDI are often included with the filler simultaneously [63], 

where others are incorporated as treatments for the filler prior to inclusion with the polymer [66]. 

Regardless of the mechanism, most commercial coupling agents are revered for their notable 

ability to improve the mechanical properties of plastics involving fillers from all sources [61-66].  

Commercial coupling agents are used in industry and are attributed to be crucial in the 

establishment of many specialized plastics and films today [38, 67]. The main hindrances for 

more extensive use of commercial coupling agents involve the price of incorporation due to the 

complicated mechanisms of creation, and the negative effects they have on the biodegradation of 

the plastics, often extending the time it takes for them to degrade naturally [61, 62]. This is 

largely attributed to the chemical nature of these compounds, and the complex interactions they 

elicit in materials affected by them, increasing the difficulty to reach groups necessary to 

decompose [61, 62]. 

2.2.4.2 Naturally Derived Coupling Agents 

There have been scarce studies investigating the potential for producing and 

characterizing a naturally occurring coupling agent. Some existing examples of such natural 

coupling agents include derivations from pine rosin [67], and palm [68] and sesame [69] oils. 

There have also been investigations into various fatty acids, including hexanoic, octanoic, 

decanoic, and dodecanoic acids [70]. However, these studies have shown minimal mechanical 

improvements, and the complex processes required limit the potential for adoption and 

application at higher production scales. 

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2.3 Soy & Soybean Meal 

Soybeans are one of the most grown crops in the world. Global production of soy reached 

more than 370 million tons in 2021 [71]. It is estimated that over 85% of this production is for 

the extraction of oil from the soybean [72]. The oil extracted is most often used for cooking oil 

production, or as biofuel [73, 74]. During this oil extraction process, soybeans are subject to hull 

cracking, removal, and separation under high heat and pressure. The exposed beans are then 

exposed to repeated hexane baths where a majority of the lipid contents are removed and isolated 

from the solid matter. The solid soybean waste that is left over is then dried, washed and 

“roasted” to produce a protein and carbohydrate rich soybean derivative deemed “soymeal”. In 

the United States, over 97% of this soymeal is sold to feed livestock, mainly cattle, swine, 

poultry, and fish, with the remainder used for protein isolate or soymilk [75]. This byproduct of 

oil extraction has a number of significant compounds within it that can be extremely beneficial 

outside animal feed. The proper extraction and utilization of these compounds can work to 

improve their investigation in science and provide increased profitability for both soybean 

farmers and refiners. 

2.3.1 Soyasaponins 

Saponins are characterized as triterpene glycosides naturally occurring in many plant 

species, particularly soapwort, licorice, and soy [76, 77]. Soyasaponins are the saponin types 

occurring naturally in soy. Soymeal has been found to contain anywhere from 0.5-1.5 wt.% 

saponin [78, 79]. There are over 10 different types of soyasaponin, with Soyasaponin I (Bb) 

being the most prevalent [80, 81]. Soyasaponins are chemically identified by the glycosylation 

sites on the backbone and characteristic surfactant properties. The basic components of their 

structure, containing both hydrophilic and hydrophobic “ends” give them these properties [82].  

Beyond this, soyasaponins have been investigated to contain numerous benefits in the 

pharmaceutical industry, including anti-cancer, antioxidant, anti-microbial, anti-inflammatory, 

and cholesterol inhibiting properties [83-86]. There has been minimal investigation for a purpose 

for soyasaponins in material science, however their unique structure gives them much potential 

for a wide variety of purposes. One study investigated saponins derived from soapberry to show 

minute improvements in tensile, flexural, and impact strength when used to treat cellulose 

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nanocrystals intended for incorporation into polypropylene in comparison to unmodified 

nanocrystal composites [87]. 

2.3.2 Soy Isoflavones 

Isoflavones are a group of phenolic compounds found in numbers across a variety of 

plant and fungal species [88]. The main isoflavone present in soy is genistein, comprising over 

60% of the total isoflavone content, the remainder of the content consists of daidzein and 

glycitein [89, 90]. The total content of soy isoflavones in defatted soymeal is recorded to be 0.2-

0.5 wt.% [89]. Soy isoflavones, when separated from the meal have also been cited to have a 

number of anti-inflammatory, anti-cancer, antioxidant, and antimicrobial benefits in medical 

settings [88].  

2.3.3 Soymeal as a Food Source 

Soymeal is one of the most popular feed choices for livestock such as chickens, 

ruminants (cattle, goats, sheep), swine, and fish [91]. Its high protein and digestible amino acid 

content (49-53 wt.%) make it extremely effective for livestock to build muscle, and its high 

carbohydrate fraction (35-39%) give it exceptional qualities as a rich energy source [92]. Despite 

its proficiency as a quality food source, there are various components of soymeal that can induce 

negative effects in some of the animals consuming it, notably, soyasaponins and soy isoflavones. 

2.3.4 Nutritional Effect of Saponins & Isoflavones in Soymeal  

Despite their extensive potential in a medical environment, soyasaponins and 

isoflavones’ presence in soymeal can cause a number of negative effects on the meal itself, and 

on some of the animals consuming it. The effects of this on the meal quality, and on livestock 

that consume the meal most are individually listed below. 

2.3.4.1 Soyasaponin & Isoflavone Effect on Flavor of Meal 

Both saponins and soy isoflavones are cited in literature for the characteristic 

overpowering bitter taste they bring to products containing them. Saponins and isoflavones from 

food sources other than soybean meal have been known to cause undesirable taste effects on 

their food products, decreasing their appeal for consumption [93-96]. Most mammals, including 

cattle and swine, have evolved to despise bitterness for its common indication of toxicity in food 

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products. Mammals have been observed to prefer food sources void of these compounds for taste 

alone [97-99]. Chickens exhibit the same, but a less pronounced effect, and fish have not shown 

any preference on taste [100, 101]. Essentially, the removal of these compounds should improve 

the desirability of the meal as a food source for cattle, swine, and chickens, and have little to no 

effect on it for fish. A meal that is more appetizing for animals will result in them eating more of 

it, inducing greater growth and production capacity for them. 

2.3.4.2 Soyasaponin & Isoflavone Effect on Health of Livestock 

Soyasaponins and isoflavones have been investigated extensively on their nutritional 

benefit to the animals that consume them. The removal of them from a soymeal product should 

produce a positive effect on the overall health, productivity, and hence profitability for cattle and 

fish in particular.  

2.3.4.2.1 Soyasaponin & Isoflavone Effect on Cattle 

Soyasaponins have been found responsible for reductions in fertility and reproductive 

health, degradation of cell membranes and intestinal linings, reduced digestion, and reduced 

appetite for cattle [102-105]. 

Soya isoflavones have been associated with early embryonic mortality or complications 

requiring abortions for cattle. Furthermore, they have been observed to disrupt the function of the 

corpus luteum, inhibiting progesterone secretion and causing additional disorders during early 

pregnancy, including early embryonic death [106-108]. 

A soymeal void of both these compounds would likely improve the metabolic health of 

cattle in comparison to the existing soymeal. 

2.3.4.2.2 Soyasaponin & Isoflavone Effect on Fish 

There have been no positive attributes to a meal higher in saponin content in the diet of 

farmed fish. Soyasaponins were determined accountable for inducing acute enteritis and severely 

decreasing digestibility in salmon, and significant intestinal ruptures in trout [109-111]. 

Isoflavones in feed have been heavily correlated to inducing skewed sex reversals in fish 

that undergo them, providing significantly more female than male when fed with meal containing 

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the isoflavones [112]. They have also been cited to disrupt reproductive systems of multiple 

species of fish [113, 114]. 

Most species of farmed fish have shown a consistent inability to digest both soyasaponins 

and soy isoflavones. Due to this, the only effects observed from these compounds are due to their 

unique structures remaining intact through the body, binding to, disturbing, and rupturing tissue 

in the digestive and reproductive systems for these animals. 

2.3.4.2.3 Soyasaponin & Isoflavone Effect on Swine 

The effects of soyasaponin content in soymeal for pigs has not been extensively studied. 

The few papers that exist suggest little to no negative effects of their presence [115] and have 

even been attributed to improving immunological responses in pigs that consume feed with them 

[116]. 

Soy isoflavones have also been significantly associated with increased immune responses 

for swine [117, 118]. However, there are correlations with isoflavones to inhibited growth and 

development for piglets, and reproductive tissue damage for pregnant or weening adult swine 

[119]. 

The complete removal of both of these compounds for meal intended as feed for swine 

may not be advised due to the significant positive effects they can have on the animal’s immune 

system.   

2.3.4.2.4 Soyasaponin & Isoflavone Effect on Poultry 

Soyasaponins in the diet of chickens have been explicitly connected to improvements in 

reproductive efficiency and overall health, with minimal negative effects [120, 121]. 

Soy Isoflavones have also been convincingly correlated to improvements in growth, 

antioxidant activity in tissue, egg laying production and quality, and improved immune 

functionality [122, 123]. 

A meal void of soyasaponins and isoflavones is not recommended in the diet of chickens 

based on the current literature. Soy isoflavones in particular are extremely beneficial to overall 

health and production of these animals.  

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2.3.5 Consequences of Removal of Soyasaponins & Isoflavones from Soymeal 

Due to the mixed effects these compounds have depending on the type of animal 

ingesting the meal, it is difficult to assess their true value. For meal that is sold to feed cattle or 

fish, soyasaponins and isoflavones can be considered a waste component that can be removed 

and used for an alternative, more beneficial purpose. The removal of these compounds from the 

meal would even produce a more desirable meal for these animals, and a decreased risk of the 

health symptoms associated with their presence, resulting in a win-win scenario.  

However, for meal being sold as feed for chickens or swine, the removal of these 

compounds can alter the state of the food source in a negative fashion. Due to the various 

positive attributes these compounds are associated with having in both these animals’ diets, they 

could be considered essential for optimal growth, health, and productivity and thus not 

acceptable for removal.  

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REFERENCES 

1. 
Jamshidian, M., Tehrany, E. A., Imran, M., Jacquot, M., & Desobry, S. Poly‐lactic acid: 
Production, applications, nanocomposites, and release studies. Comprehensive Reviews in Food 
Science and Food Safety, 9(5), pp 552–571 (2010).   

Mehta, R., Kumar, V., Bhunia, H., & Upadhyay, S. N.  Synthesis of poly(lactic acid): A 

2. 
Review. Journal of Macromolecular Science, Part C: Polymer Reviews, 45(4), pp 325–349. 
(2005) https://doi.org/10.1080/15321790500304148  

3. 
Lemos Machado Abreu, A. S., de Moura, I. G., de Sá, A. V., & Alves Machado, A. V., 
Biodegradable polymer nanocomposites for packaging applications. Food Packaging, pp 329–
363. (2017) https://doi.org/10.1016/b978-0-12-804302-8.00010-8  

Byun, Y., & Kim, Y. T. Bioplastics for Food Packaging. Innovations in Food Packaging, 

4. 
pp 353–368 (2014). https://doi.org/10.1016/b978-0-12-394601-0.00014x 

Classification of polymer additives. Introduction of Polymer additives | Chemical 

5. 
Products｜ADEKA.  

Ebnesajjad, S. Effect of chemicals on plastics. Chemical Resistance of Engineering 

6. 
Thermoplastics, pp 27–41. (2016) https://doi.org/10.1016/b978-0-323-47357-6.00022-2  

Niaounakis, M. (2013). Introduction to biopolymers. Biopolymers Reuse, Recycling, and 

7. 
Disposal, 1–75. https://doi.org/10.1016/b978-1-4557-3145-9.00001-4  

8. 

Light stabilisers. ADEKA Polymer Additives Europe. (2014).   

Antioxidants polymer additives: The effective oxidation fighters: Tosaf - additive and 

9. 
color solutions for the plastic industry. Tosaf. (2022, June 15).   

10. 
Duguma, H. T., Khule, P., McArdle, A., Fennell, K., & Almenar, E., Turning agricultural 
waste into packages for food: A literature review from origin to end-of-life. Food Packaging and 
Shelf Life, 40, 101166. (2023) https://doi.org/10.1016/j.fpsl.2023.101166  

11. 
Chowdhury, A.,  Sarkar, A., Chapter 30 -  Vermicomposting-the sustainable solid waste 
management, Waste Management and Resource Recycling in the Developing World pp 701-719 
(2023) doi:10.1016/B978-0.323-90463-6.00013-0 

La Mantia, F. P., & Morreale, M. Green composites: A brief review. Composites Part A: 

12. 
Applied Science and Manufacturing, 42(6), 579–588. (2011). 
https://doi.org/10.1016/j.compositesa.2011.01.017 

13.  McGauran, T., Dunne, N., Smyth, B. M., & Cunningham, E. Incorporation of poultry 
eggshell and litter ash as high loading polymer fillers in polypropylene. Composites Part C: 
100080(2020). https://doi.org/10.1016/j.jcomc.2020.100080 

14. 
Tsou, C.-H., Chen, Z.-J., Yuan, S., Ma, Z.-L., Wu, C.-S., Yang, T., Jia, C.-F., & Reyes 
De Guzman, M. The preparation and performance of poly(butylene adipate) terephthalate/corn 
stalk composites. Current Research in Green and Sustainable Chemistry, 5, 100329. (2022) 
https://doi.org/10.1016/j.crgsc.2022.100329 

22 
22 

 
 
 
 
 
 
Fehlberg, J., McKay, S., Matuana, L. M., & Almenar, E. Use of orange juice processing 

15. 
waste to produce films using blown film extrusion for food packaging. Journal of Food 
Engineering, 341, 111337 (2023). https://doi.org/10.1016/j.jfoodeng.2022.111337 

Gustafsson, J., Landberd, M., Production of Bio-Plastic Materials From Apple Pomace 

16. 
(2018) 

Sánchez-Safont, E. L., Aldureid, A., Lagarón, J. M., Gámez-Pérez, J., & Cabedo, L. 

17. 
Biocomposites of different lignocellulosic wastes for Sustainable Food Packaging Applications. 
Composites Part B: Engineering, 145, 215–225. (2018). 
https://doi.org/10.1016/j.compositesb.2018.03.037 

Suaduang, N., Ross, S., Ross, G. M., Pratumshat, S., & Mahasaranon, S., Effect of spent 

18. 
coffee grounds filler on the physical and mechanical properties of poly(lactic acid) bio-
composite films. Materials Today: Proceedings, 17, 2104–2110 (2019). 
https://doi.org/10.1016/j.matpr.2019.06.260 

19. 
Alam Faroque, F., Bandhu Ghosh, S., & Bandyopadhyay-Ghosh, S. Effect of chemical 
treatment on mechanical properties and water resistance of wheat straw epoxy biocomposites. 
Materials Today: Proceedings, 58, 761–763. (2022). https://doi.org/10.1016/j.matpr.2022.02.577 

Terzioğlu, P., Güney, F., Parın, F. N., Şen, İ., & Tuna, S. Biowaste Orange Peel 

20. 
Incorporated Chitosan/polyvinyl alcohol composite films for Food Packaging Applications. Food 
Packaging and Shelf Life, 30, 100742 (2021). https://doi.org/10.1016/j.fpsl.2021.100742 

21. 
Lima, E. M., Middea, A., Neumann, R., Thiré, R. M., Pereira, J. F., de Freitas, S. C., 
Penteado, M. S., Lima, A. M., Minguita, A. P., Mattos, M. da, Teixeira, A. da, Pereira, I. C., 
Rojas dos Santos, N. R., Marconcini, J. M., Oliveira, R. N., & Corrêa, A. C. Biocomposites of 
PLA and mango seed waste: Potential material for food packaging and a technological 
alternative to reduce environmental impact. Starch - Stärke, 73(5–6). (2021) 
https://doi.org/10.1002/star.202000118 

La Mantia, F., Dintcheva, N. T., Morreale, M., & Vaca‐Garcia, C. Green composites of 

22. 
organic materials and recycled post‐consumer polyethylene. Polymer International, 53(11), 
1888–1891. (2004) https://doi.org/10.1002/pi.1614 

23.  Meereboer, K.W., Pal, A.K., Cisneros-López, E.O. et al. The effect of natural fillers on 
the marine biodegradation behaviour of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). 
Sci Rep 11, 911 (2021). https://doi.org/10.1038/s41598-020-78122-7 

Kumar, R., Yakubu, M. K., & Anandjiwala, R. D. Biodegradation of flax fiber reinforced 

24. 
poly lactic acid. Express Polymer Letters, 4(7), 423–430. (2010) 
https://doi.org/10.3144/expresspolymlett.2010.53 

Ashori, A. Hybrid thermoplastic composites using nonwood plant fibers. Hybrid Polymer 

25. 
Composite Materials, 39–56 (2017) https://doi.org/10.1016/b978-0-08-100787-7.00002-0 

Hamzeh, Y., Ashori, A., Marvast, E. H., Rashedi, K., & Olfat, A. M. A comparative 

26. 
study on the effects of coriolus versicolor on properties of HDPE/wood flour/paper sludge 
composites. Composites Part B: Engineering, 43(5), 2409–2414. (2012) 
https://doi.org/10.1016/j.compositesb.2011.11.043 

23 
23 

 
 
 
 
 
 
Top 4 reasons calcium carbonate is used by plastic manufacturers as fillers " Daia 

27. 
industry. Daia Industry. (2023, June 22). 

28. 
Premphet, K., & Horanont, P. Improving performance of polypropylene through 
combined use of calcium carbonate and metallocene-produced impact modifier. Polymer-Plastics 
Technology and Engineering, 40(3), 235–247. (2001) https://doi.org/10.1081/ppt-100000245 

29.  Wang, K., Bahlouli, N., Addiego, F., Ahzi, S., Rémond, Y., Ruch, D., & Muller, R. 
Effect of talc content on the degradation of re-extruded polypropylene/talc composites. Polymer 
Degradation and Stability, 98(7), 1275–1286. (2013) 
https://doi.org/10.1016/j.polymdegradstab.2013.04.006 

30. 
Oliver-Ortega, H., Tresserras, J., Julian, F., Alcalà, M., Bala, A., Espinach, F. X., & 
Méndez, J. A. Nanocomposites materials of PLA reinforced with Nanoclays using a masterbatch 
technology: A study of the mechanical performance and its sustainability. Polymers, 13(13), 
2133. (2021)  https://doi.org/10.3390/polym13132133 

31. 
Helanto, K., Talja, R., & Rojas, O. J. (2021). Effects of talc, kaolin and calcium 
carbonate as fillers in biopolymer packaging materials. Journal of Polymer Engineering, 41(9), 
746–758. https://doi.org/10.1515/polyeng-2021-0076 

Siročić, A. P., Rešček, A., Ščetar, M., Krehula, L. K., & Hrnjak-Murgić, Z. (2013). 

32. 
Development of low density polyethylene nanocomposites films for packaging. Polymer 
Bulletin, 71(3), 705–717. https://doi.org/10.1007/s00289-013-1087-9 

Delgado, K., Quijada, R., Palma, R., & Palza, H. (2011). Polypropylene with embedded 

33. 
copper metal or copper oxide nanoparticles as a novel plastic antimicrobial agent. Letters in 
Applied Microbiology, 53(1), 50–54. https://doi.org/10.1111/j.1472-765x.2011.03069.x 

Bazant, P., Kuritka, I., Munster, L., Machovsky, M., Kozakova, Z., & Saha, P. (2014). 

34. 
Hybrid nanostructured ag/zno decorated powder cellulose fillers for medical plastics with 
enhanced surface antibacterial activity. Journal of Materials Science: Materials in Medicine, 
25(11), 2501–2512. https://doi.org/10.1007/s10856-014-5274-5 

Aframehr, W. M., Molki, B., Heidarian, P., Behzad, T., Sadeghi, M., & Bagheri, R. 

35. 
(2017). Effect of calcium carbonate nanoparticles on barrier properties and biodegradability of 
polylactic acid. Fibers and Polymers, 18(11), 2041–2048. https://doi.org/10.1007/s12221-017-
6853-0 

36. 
Diao, X., Zhang, C., & Weng, Y. (2022). Properties and degradability of poly(butylene 
adipate-co-terephthalate)/calcium carbonate films modified by polyethylene glycol. Polymers, 
14(3), 484. https://doi.org/10.3390/polym14030484 

37. 

Rissi, G. O. (2020, July 21). Fillers for packaging applications. IntechOpen. 

Coleman, E. A. (2011). Plastics additives. Applied Plastics Engineering Handbook, 419–

38. 
428. https://doi.org/10.1016/b978-1-4377-3514-7.10023-6 

Semakina, O. K., Phomenko, A. N., Leonteva, A. A., & Rymanova, I. E. (2015). 

39. 
Research of surface properties of fillers for polymers. Procedia Chemistry, 15, 79–83. 
https://doi.org/10.1016/j.proche.2015.10.012 

24 
24 

 
 
 
 
 
 
40. 
Khoshkava, V., & Kamal, M. R. (2014). Effect of drying conditions on cellulose 
nanocrystal (CNC) agglomerate porosity and dispersibility in polymer nanocomposites. Powder 
Technology, 261, 288–298. https://doi.org/10.1016/j.powtec.2014.04.016 

Chen, F., Shi, W., Zhou, D., Zhang, Q., Lv, L., Sun, J., & Yang, L. (2018). 

41. 
Nanostructured thin lignin-derived carbon sheets as excellent reinforcement fillers in 
polypropylene. RSC Advances, 8(65), 37472–37479. https://doi.org/10.1039/c8ra08302a 

42.  Wang, Lu, "Spray-Dried Cellulose Nanofibril-Reinforced Polypropylene Composites for 
Extrusion-Based Additive Manufacturing" (2017). University of Maine Electronic Theses and 
Dissertations. 2791. 

43. 
Candido, R. G., Godoy, G. G., & Gonçalves, A. R. (2017). Characterization and 
application of cellulose acetate synthesized from sugarcane bagasse. Carbohydrate Polymers, 
167, 280–289. https://doi.org/10.1016/j.carbpol.2017.03.057 

Syuhada, D. N., & Azura, A. R. (2021). Waste natural polymers as potential fillers for 

44. 
biodegradable latex-based composites: A Review. Polymers, 13(20), 3600. 
https://doi.org/10.3390/polym13203600 

Freitas, P. A. V., González-Martínez, C., & Chiralt, A. (2022). Applying ultrasound-

45. 
assisted processing to obtain cellulose fibres from rice straw to be used as reinforcing agents. 
Innovative Food Science & Emerging Technologies, 76, 102932. 
https://doi.org/10.1016/j.ifset.2022.102932 

46. 
Prabhu, L., Krishnaraj, V., Gokulkumar, S., Sathish, S., & Ramesh, M. (2019). 
Mechanical, chemical and acoustical behavior of sisal – tea waste – glass fiber reinforced epoxy 
based hybrid polymer composites. Materials Today: Proceedings, 16, 653–660. 
https://doi.org/10.1016/j.matpr.2019.05.142 

Khan, M. N., Rehman, N., Sharif, A., Ahmed, E., Farooqi, Z. H., & Din, M. I. (2020). 

47. 
Environmentally benign extraction of cellulose from Dunchi fiber for nanocellulose fabrication. 
International Journal of Biological Macromolecules, 153, 72–78. 
https://doi.org/10.1016/j.ijbiomac.2020.02.333 

SÖĞÜT, E., & SEYDİM, A. C. (2022). Utilization of kiwi peel lignocellulose as fillers in 

48. 
poly(lactic acid) films. Journal of the Turkish Chemical Society Section A: Chemistry, 9(1), 
283–294. https://doi.org/10.18596/jotcsa.1024326 

49. 
Zhou, L., Ke, K., Yang, M.-B., & Yang, W. (2021). Recent progress on chemical 
modification of cellulose for high mechanical-performance poly(lactic acid)/cellulose composite: 
A Review. Composites Communications, 23, 100548. 
https://doi.org/10.1016/j.coco.2020.100548 

Cudjoe, E., Hunsen, M., Xue, Z., Way, A. E., Barrios, E., Olson, R. A., Hore, M. J. A., & 

50. 
Rowan, S. J. (2017). Miscanthus giganteus: A commercially viable sustainable source of 
cellulose nanocrystals. Carbohydrate Polymers, 155, 230–241. 
https://doi.org/10.1016/j.carbpol.2016.08.049 

51. 
Xie, H., Zou, Z., Du, H., Zhang, X., Wang, X., Yang, X., Wang, H., Li, G., Li, L., & Si, 
C. (2019). Preparation of thermally stable and surface-functionalized cellulose nanocrystals via 
mixed H2SO4/oxalic acid hydrolysis. Carbohydrate Polymers, 223, 115116. 
https://doi.org/10.1016/j.carbpol.2019.115116 

25 
25 

 
 
 
 
 
 
52. 
Teow, Y. H., Amirudin, S. N., & Ho, K. C. (2020). Sustainable approach to the synthesis 
of cellulose membrane from oil palm empty fruit bunch for dye wastewater treatment. Journal of 
Water Process Engineering, 34, 101182. https://doi.org/10.1016/j.jwpe.2020.101182 

Bellili, N., Dairi, B., Hammour, N., Djidjelli, H., & Boukerrou, A. (2022). 

53. 
Physicomechanical behavior of agro waste-filled polypropylene composites materials. Materials 
Today: Proceedings, 53, 42–45. https://doi.org/10.1016/j.matpr.2021.12.259 

54.  Martelli-Tosi, M., Masson, M. M., Silva, N. C., Esposto, B. S., Barros, T. T., Assis, O. B. 
G., & Tapia-Blácido, D. R. (2018). Soybean straw nanocellulose produced by enzymatic or acid 
treatment as a reinforcing filler in soy protein isolate films. Carbohydrate Polymers, 198, 61–68. 
https://doi.org/10.1016/j.carbpol.2018.06.053 

Leskovac, M., Kovačević, V., Lučić Blagojević, S., Vrsaljko, D., & Volovšek, V. (2004). 

55. 
Pre-treatment of CACO3 nanofiller by irradiation in the presence of vinyl monomers for the 
preparation of poly(vinyl acetate) composites. E-Polymers, 4(1). 
https://doi.org/10.1515/epoly.2004.4.1.348 

Ippolito, F., Hübner, G., Claypole, T., & Gane, P. (2020). Influence of the surface 

56. 
modification of calcium carbonate on polyamide 12 composites. Polymers, 12(6), 1295. 
https://doi.org/10.3390/polym12061295 

57. 
Ahmad Fauzi, A. A., Osman, A. F., Alosime, E. M., Ibrahim, I., Abdul Halim, K. A., & 
Ismail, H. (2022). Strategies towards producing non-polar dolomite nanoparticles as nanofiller 
for copolymer nanocomposite. International Journal of Molecular Sciences, 23(20), 12620. 
https://doi.org/10.3390/ijms232012620 

Leow, V. J., Teh, P. L., Yeoh, C. K., Abdul Rahim, N. A., Wong, W. C., Voon, C. H., 

58. 
Mohamed Rasidi, M. S., & Lim, B. Y. (2023). The effect of coated calcium carbonate using 
stearic acid on the recovered carbon black masterbatch in low-density polyethylene composites. 
E-Polymers, 23(1). https://doi.org/10.1515/epoly-2023-0025 

Zhao, Z., Guo, Q., Qian, J., & Pan, G. (2012). Mechanical properties and tribological 

59. 
behaviour of polyurethane elastomer reinforced with Caco3 Nanoparticles. Polymers and 
Polymer Composites, 20(7), 575–580. https://doi.org/10.1177/096739111202000701 

Understanding coupling agents. Fraunhofer Institute for Structural Durability and System 

60. 
Reliability LBF. (2023, August 2). 

Rakmae, S., Ruksakulpiwat, Y., Sutapun, W., & Suppakarn, N. (2012). Effect of silane 

61. 
coupling agent treated bovine bone based carbonated hydroxyapatite on in vitro degradation 
behavior and bioactivity of PLA Composites. Materials Science and Engineering: C, 32(6), 
1428–1436. https://doi.org/10.1016/j.msec.2012.04.022 

Roumeli, E., Terzopoulou, Z., Pavlidou, E., Chrissafis, K., Papadopoulou, E., 

62. 
Athanasiadou, E., Triantafyllidis, K., & Bikiaris, D. N. (2015). Effect of maleic anhydride on the 
mechanical and thermal properties of hemp/high-density polyethylene green composites. Journal 
of Thermal Analysis and Calorimetry, 121(1), 93–105. https://doi.org/10.1007/s10973-015-4596-
y 

Baek, B.-S., Park, J.-W., Lee, B.-H., & Kim, H.-J. (2013). Development and application 

63. 
of Green Composites: Using coffee ground and Bamboo Flour. Journal of Polymers and the 
Environment, 21(3), 702–709. https://doi.org/10.1007/s10924-013-0581-3 

26 
26 

 
 
 
 
 
 
64.  Monte, S. J., Sugerman, G., Damusis, A., & Patel, P. (1982). Application of Titanate 
Coupling Agents in Mineral and Glass Fiber Filled RIM Urethane Systems. SAE Transactions, 
91, 1702–1724. http://www.jstor.org/stable/44724632 

65. 
Leduc, S., Ureña, J. R., González-Núñez, R., Quirarte, J. R., Riedl, B., & Rodrigue, D. 
(2008). LDPE/Agave fibre composites: Effect of coupling agent and weld line on mechanical 
and morphological properties. Polymers and Polymer Composites, 16(2), 115–123. 
https://doi.org/10.1177/096739110801600204 

Lučić Blagojević, Vera Kovačević, Mirela Leskovac, Domagoj Vrsaljko, Vesna 

66. 
Volovšek, Christoph Nover, Silane pre-treatment of calcium carbonate nanofillers for 
polyurethane composites Sanja   Faculty of Chemical Engineering and Technology, University 
of Zagreb, Marulićev trg 19,10000 Zagreb, Croatia; Fax +385 1 4597 260; slucic@marie.fkit.hr 
2 Solvay Co., Ludwigstr. 10, 47495 Rheinberg, Germany 

Inga-Lafebre, J., Pulido-González, H., González-Núñez, R., Hernández-Hernández, M. 

67. 
E., Rabelero-Velasco, M., Aranda-García, F. J., & Jasso-Gastinel, C. F. (2019). The multirole of 
modified natural gums for multicomponent polymers: As coupling agents for polymers 
reinforced with cellulosic fibers or compatibilizers for biodegradable polymer blends. Química 
Nova. https://doi.org/10.21577/0100-4042.20170333 

KoaySeong Chun, SalmahHusseinsyah, Hakimah Osman, Preliminary Study of Palm Oil 

68. 
Based Coupling Agent forPolypropylene/Cocoa Pod Husk Composites. Adv. Environ. Biol., 
8(8), 2640-2644, (2014) 

R. Ortega-Toro, A. López-Córdoba, F. Avalos-Belmontes, Epoxidised sesame oil as a 

69. 
biobased coupling agent and plasticizer in polylactic acid/thermoplastic yam starch blends, 
Heliyon (2021) doi:10.1016/j.heliyon.2021.e06176 

Poletto M., et al., Natural Oils as coupling agents in recycled polypropylene wood flour 

70. 
composites: Mechanical, thermal and morphological properties, Polymers and Polymer 
Composites (2019), doi:10.1177/0967391119886941 

71.  Wunsch, N.-G. (2023, August 24). Most grown crops in the world 2021. Statista. 

Cooking oil market - share, Trends & Industry Overview. Cooking Oil Market - Share, 

72. 
Trends & Industry Overview. (2023) 

Khan, M., Bonifacio, S., Clowes, J., Foulds, A., Holland, R., et al., Investigation of 

73. 
Biofuel as a Potential Renewable Energy Source, Air Quality Vol. 12 Issue 10 
doi:10.3390/atmos12101289 

Jain, V.,  Proctor, A., 12 – CLA Production by Photo-isomerization of Linoleic Acid in 

74. 
Linoleic Acid Rich Oils, Green Vegetable Oil Processing 1st Edition pp 225-263, 
doi:10.1016/B978-0-9888565-3-0.50015-7 

Amol, V., Bhati, K., A Literature Review on effects and uses of compounds in soybean, 

75. 
JETIR Vol. 9 Issue 1 ISSN:2349-5162  

Vincken, J.-P., Heng, L., de Groot, A., & Gruppen, H. (2007). Saponins, classification 

76. 
and occurrence in the plant kingdom. Phytochemistry, 68(3), 275–297. 
https://doi.org/10.1016/j.phytochem.2006.10.008 

27 
27 

 
 
 
 
 
 
77.  Mugford, S. T., & Osbourn, A. (2012). Saponin synthesis and function. Isoprenoid 
Synthesis in Plants and Microorganisms, 405–424. https://doi.org/10.1007/978-1-4614-4063-
5_28 

Knudsen, D., Røn, Ø., Baardsen, G., Smedsgaard, J., Koppe, W., & Hanne Frøkiær. 

78. 
(2006). Soyasaponins resist extrusion cooking and are not degraded during gut passage in 
Atlantic Salmon (salmo salar L.). Journal of Agricultural and Food Chemistry, 54(17), 6428–
6435. https://doi.org/10.1021/jf0604992 

79. 
Ireland, P. A., Dziedzic, S. Z., & Kearsley, M. W. (1986). Saponin content of soya and 
some commercial soya products by means of high‐performance liquid chromatography of the 
sapogenins. Journal of the Science of Food and Agriculture, 37(7), 694–698. 
https://doi.org/10.1002/jsfa.2740370715 

Kamo, S., Suzuki, S., & Sato, T. (2014). The content of soyasaponin and soyasapogenol 

80. 
in soy foods and their estimated intake in the Japanese. Food Science & Nutrition, 2(3), 
289–297. https://doi.org/10.1002/fsn3.107 

Zhang, W., & Popovich, D. (2009). Chemical and biological characterization of Oleanane 

81. 
triterpenoids from soy. Molecules, 14(8), 2959–2975. 
https://doi.org/10.3390/molecules14082959 

82.  Moses, T., Papadopoulou, K. K., & Osbourn, A. (2014). Metabolic and functional 
diversity of saponins, biosynthetic intermediates and semi-synthetic derivatives. Critical Reviews 
in Biochemistry and Molecular Biology, 49(6), 439–462. 
https://doi.org/10.3109/10409238.2014.953628 

Omar, A., Kalra, R. S., Putri, J., Elwakeel, A., Kaul, S. C., & Wadhwa, R. (2020). 

83. 
Soyasapogenol-a targets carf and results in suppression of tumor growth and metastasis in p53 
compromised cancer cells. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-62953-
5 

Chen, J., Ullah, H., Zheng, Z., Gu, X., Su, C., Xiao, L., Wu, X., Xiong, F., Li, Q., & Zha, 

84. 
L. (2020). Soyasaponins reduce inflammation by downregulating MyD88 expression and 
suppressing the recruitments of TLR4 and myd88 into lipid rafts. BMC Complementary 
Medicine and Therapies, 20(1). https://doi.org/10.1186/s12906-020-2864-2 

85. 
Guang, C., Chen, J., Sang, S., & Cheng, S. (2014). Biological functionality of 
Soyasaponins and Soyasapogenols. Journal of Agricultural and Food Chemistry, 62(33), 8247–
8255. https://doi.org/10.1021/jf503047a 

Yoon, G.-A., & Park, S. (2014). Antioxidant action of soy isoflavones on oxidative stress 

86. 
and antioxidant enzyme activities in exercised rats. Nutrition Research and Practice, 8(6), 618. 
https://doi.org/10.4162/nrp.2014.8.6.618 

Cherian, B. M., Leão, A. L., Caldeira, M. da, Chiarelli, D., de Souza, S. F., Narine, S., & 

87. 
de Morais Chaves, M. R. (2012). Use of Saponins as an effective surface modifier in cellulose 
nanocomposites. Molecular Crystals and Liquid Crystals, 556(1), 233–245. 
https://doi.org/10.1080/15421406.2012.635969 

Yu, J., Bi, X., Yu, B., & Chen, D. (2016). Isoflavones: Anti-inflammatory benefit and 

88. 
possible caveats. Nutrients, 8(6), 361. https://doi.org/10.3390/nu8060361 

28 
28 

 
 
 
 
 
 
USDA database for the isoflavone content of selected foods, release 2. USDA DataBase 

89. 
for the Isoflavone Content of Selected Foods. (2008, September). 

Fukutake, M., Takahashi, M., Ishida, K., Kawamura, H., Sugimura, T., & Wakabayashi, 

90. 
K. (1996). Quantification of genistein and Genistin in soybeans and soybean products. 

Pantalone, V., 11 – Modern Breeding Approaches to Enhancing Soy Protein Quality, 

91. 
Designing Soybeans for 21st Century Markets pp 197-226 (2012) doi:10.1016/B978-0-9830791-
0-1.50015-7 

92. 
Toomer, O. T., Oviedo, E. O., Ali, M., Patino, D., et al., Cuurent Agronomic Practices, 
Harvest & Post-Harvest Processing of Soybeans (Glycine max)- A Review, Agronomy Vol 13 
pp 427 (2023) doi:10.3390/agronomy13020427 

Spagnuolo, C., Russo, G. L., Orhan, I. E., Habtemariam, S., Daglia, M., Sureda, A., 

93. 
Nabavi, S. F., Devi, K. P., Loizzo, M. R., Tundis, R., & Nabavi, S. M. (2015). Genistein and 
cancer: Current status, challenges, and Future Directions. Advances in Nutrition, 6(4), 408–419. 
https://doi.org/10.3945/an.114.008052 

Drewnowski, A., & Gomez-Carneros, C. (2000). Bitter taste, phytonutrients, and the 

94. 
consumer: A Review. The American Journal of Clinical Nutrition, 72(6), 1424–1435. 
https://doi.org/10.1093/ajcn/72.6.1424 

95.  Mikołajczyk-Bator, K. (2022). The significance of Saponins in shaping the quality of 
food products from Red Beet. Acta Scientiarum Polonorum Technologia Alimentaria, 21(1), 81–
90. https://doi.org/10.17306/j.afs.1012 

96. 
Dawid, C., & Hofmann, T. (2014). Quantitation and bitter taste contribution of saponins 
in fresh and cooked white asparagus (asparagus officinalis L.). Food Chemistry, 145, 427–436. 
https://doi.org/10.1016/j.foodchem.2013.08.057 

Barnes, A. (2022, September 6). Cattle: What you need to know. The Open Sanctuary 

97. 
Project.  

98.  McLaughlin, C. L., Baile, C. A., Buckholtz, L. L., & Freeman, S. K. (1983). Preferred 
flavors and performance of Weanling Pigs. Journal of Animal Science, 56(6), 1287–1293. 
https://doi.org/10.2527/jas1983.5661287x 

Forbes M. 1995. Voluntary Food Intake and Diet Selection in Farm Animals. 

99. 
Walingford: CABI. p245. 

100.  Kasumyan, A. O. (2019). The taste system in fishes and the effects of environmental 
variables. Journal of Fish Biology, 95(1), 155–178. https://doi.org/10.1111/jfb.13940 

101.  Liu, H.-X., Rajapaksha, P., Wang, Z., E Kramer, N., & J Marshall, B. (2018). An update 
on the sense of taste in chickens: A better developed system than previously appreciated. Journal 
of Nutrition & Food Sciences, 08(02). https://doi.org/10.4172/2155-9600.1000686 

102.  Grgic, D., Varga, E., Novak, B., Müller, A., & Marko, D. (2021). Isoflavones in animals: 
Metabolism and effects in livestock and occurrence in feed. Toxins, 13(12), 836. 
https://doi.org/10.3390/toxins13120836 

103.  Szkudelska, K., Szumacher-Strabel, M., Szczechowiak, J., Bryszak, M., Pers-Kamczyc, 
E., Stochmal, A., & Cieslak, A. (2015). The effect of triterpenoid saponins fromsaponaria 

29 
29 

 
 
 
 
 
 
officinalison some blood hormones, metabolic parameters and fatty acid composition in dairy 
cows. The Journal of Agricultural Science, 154(3), 532–541. 
https://doi.org/10.1017/s0021859615001070 

104.  Das, T., Banerjee, D., Chakraborty, D., Pakhira, M., Shrivastava, B., & Kuhad, R. (2012). 
Saponin: Role in Animal System. Veterinary World, 5(4), 248. 
https://doi.org/10.5455/vetworld.2012.248-254 

105.  Ginane, C., Baumont, R., & Favreau-Peigné, A. (2011). Perception and hedonic value of 
basic tastes in domestic ruminants. Physiology & Behavior, 104(5), 666–674. 
https://doi.org/10.1016/j.physbeh.2011.07.011 

106.  Okuda, K., Miyamoto, Y., & Skarzynski, D. J. (2002). Regulation of endometrial 
prostaglandin F2Α synthesis during luteolysis and early pregnancy in cattle. Domestic Animal 
Endocrinology, 23(1–2), 255–264. https://doi.org/10.1016/s0739-7240(02)00161-3 

107.  Wolawek-Potocka, I., Bah, M. M., Korzekwa, A., Piskula, M. K., Wiczkowski, W., 
Depta, A., & Skarzynski, D. J. (2005). Soybean-derived phytoestrogens regulate prostaglandin 
secretion in endometrium during cattle estrous cycle and early pregnancy. Experimental Biology 
and Medicine, 230(3), 189–199. https://doi.org/10.1177/153537020523000305 

108.  Shore, L. S., Rios, C., Marcus, S., Bernstein, M., & Shemesh, M. (1998). Relationship 
between peripheral estrogen concentrations at insemination and subsequent fetal loss in cattle. 
Theriogenology, 50(1), 101–107. https://doi.org/10.1016/s0093-691x(98)00117-4 

109.  Chikwati, E. M., Venold, F. F., Penn, M. H., Rohloff, J., Refstie, S., Guttvik, A., 
Hillestad, M., & Krogdahl, Å. (2011). Interaction of soyasaponins with plant ingredients in diets 
for Atlantic salmon, salmo salar L. British Journal of Nutrition, 107(11), 1570–1590. 
https://doi.org/10.1017/s0007114511004892 

110. 
Iwashita, Y., Suzuki, N., Matsunari, H., Sugita, T., & Yamamoto, T. (2009). Influence of 
soya saponin, soya lectin, and cholyltaurine supplemented to a casein-based semipurified diet on 
intestinal morphology and biliary bile status in fingerling rainbow trout Oncorhynchus mykiss. 
Fisheries Science, 75(5), 1307–1315. https://doi.org/10.1007/s12562-009-0158-1 

111.  Knudsen, D., Røn, Ø., Baardsen, G., Smedsgaard, J., Koppe, W., & Hanne Frøkiær. 
(2006). Soyasaponins resist extrusion cooking and are not degraded during gut passage in 
Atlantic Salmon (salmo salar L.). Journal of Agricultural and Food Chemistry, 54(17), 6428–
6435. https://doi.org/10.1021/jf0604992 

112.  Turan, F., Yigitarslan, K. D., (2019) Effect of immersion treatment of soybean 
isoflavones extract on sex reversal in the Rainbow Trout (Oncorhynchis mykiss, Walbaum, 
1792) Biozoo Journal Article no. e181307 

113.  Rzepkowska, M., Roszko, M. Ł., Fajkowska, M., Adamek‐Urbańska, D., & Ostaszewska, 
T. (2020). Dietary isoflavone intake and tissue concentration in cultured sturgeons. Aquaculture 
Nutrition, 26(3), 866–875. https://doi.org/10.1111/anu.13045 

114.  Pastore, M. R., Negrato, E., Poltronieri, C., Barion, G., Messina, M., Tulli, F., Ballarin, 
C., Maccatrozzo, L., Radaelli, G., & Bertotto, D. (2018). Effects of dietary soy isoflavones on 
estrogenic activity, cortisol level, health and growth in rainbow trout, oncorhynchus mykiss. 
Aquaculture Research, 49(4), 1469–1479. https://doi.org/10.1111/are.13602 

30 
30 

 
 
 
 
 
 
115.  Topping, D. L., Storer, G. B., Calvert, G. D., Illman, R. J., Oakenfull, D. G., & Weller, R. 
A. (1980). Effects of dietary saponins on fecal bile acids and neutral sterols, plasma lipids, and 
lipoprotein turnover in the pig. The American Journal of Clinical Nutrition, 33(4), 783–786. 
https://doi.org/10.1093/ajcn/33.4.783 

116.  Li, Y. P., Jiang, X. R., Wei, Z. X., Cai, L., Yin, J. D., & Li, X. L. (2020). Effects of 
soybean isoflavones on the growth performance, intestinal morphology and antioxidative 
properties in pigs. Animal, 14(11), 2262–2270. https://doi.org/10.1017/s1751731120001123 

117.  Smith, B. N., Morris, A., Oelschlager, M. L., Connor, J., & Dilger, R. N. (2019). Effects 
of dietary soy isoflavones and soy protein source on response of weanling pigs to porcine 
reproductive and respiratory syndrome viral infection. Journal of Animal Science, 97(7), 2989–
3006. https://doi.org/10.1093/jas/skz135 

118.  Cook, D. R., The Effect of Dietary Soybean Isoflavones on the Rate and Efficiency of 
Growth and Carcass Muscle Content in Pigs and Rats (1998)  

119.  Li, P., Zhao, Y., Yan, S., Song, B., Liu, Y., Gao, M., Tang, D., & Guo, Y. (2021). Soya 
saponin improves egg-laying performance and immune function of laying hens. Journal of 
Animal Science and Biotechnology, 12(1). https://doi.org/10.1186/s40104-021-00647-2 

120.  Li, P., Gao, M., Song, B., Yan, S., Zhao, Y., Gong, L., Liu, Y., Lv, Z., & Guo, Y. (2022). 
Soya saponin fails to improve the antioxidation and immune function of laying hens with 
antibiotics treated. Poultry Science, 101(8), 101921. https://doi.org/10.1016/j.psj.2022.101921 

Jiang, Z. Y., Jiang, S. Q., Lin, Y. C., Xi, P. B., Yu, D. Q., & Wu, T. X. (2007). Effects of 

121. 
soybean isoflavone on growth performance, meat quality, and Antioxidation in male broilers. 
Poultry Science, 86(7), 1356–1362. https://doi.org/10.1093/ps/86.7.1356 

122.  Azzam, M. M., JIANG, S., CHEN, J., LIN, X., GOU, Z., FAN, Q., WANG, Y., LI, L., & 
JIANG, Z. (2019). Effect of soybean isoflavones on growth performance, immune function, and 
viral protein 5 mrna expression in broiler chickens challenged with infectious bursal disease 
virus. Animals, 9(5), 247. https://doi.org/10.3390/ani9050247 

123.  Abdelghani, E., Xing, W., Li, Y., Shen, D., Alsiddig, M., & Li, C. (2019). Effects of 
dietary supplementation of soy isoflavones on the performance and egg quality in native Chinese 
breeder hens. Brazilian Journal of Poultry Science, 21(4). https://doi.org/10.1590/1806-9061-
2018-0940 

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CHAPTER 3 

MATERIALS & METHODS 

3.1 Extraction and Quantification of Saponin Derivative from Soymeal 

3.1.1 Materials 

Defatted soymeal was provided by Zeeland Farm Services (ZFS) Inc. soybean processing 

facility (Ithaca, MI, USA). N-butanol (>99% purity), food-grade ethanol (>95% purity), and 

soyasaponin standard (Soyasaponin I (Bb) of >95% purity) were all purchased from Sigma 

Aldrich (Sigma Aldrich St. Louis MO, USA) and Cayman Chemical (Cayman Chemical Ann 

Arbor MI, USA). Deionized water was supplied by the Michigan State School of Packaging. 

3.1.2 Extraction of Soyasaponin Derivative 

Following the procedures of [1], & [2], 10 g of the defatted soymeal was subject to a 24-

hour maceration in 200 mL of 60% aqueous ethanol inside 12 oz jars without agitation. Following 

the  maceration,  the  meal  was  vacuum  filtered  out  of  the  solution  using  a  no.  1  filter  paper 

(Whatman qualitative filter paper, grade 1, Cat no. 1001 110) and a GAST model DOA-P704-AA 

vacuum pump (Max Pressure 60psi, Volts: 115Amps: 4.2Hz:60) until no liquid continued to drop 

for at least 10 seconds. 

The  filtered  liquid  was  then  placed  in  an  open  250-mL  beaker  and  evaporated  at  room 

temperature  until  only  solid  or  very  viscous  residue  deemed  “soybean  molasses”  remained. 

Following the procedure of [3], the dry residue was introduced to a 1:1 (v/v) mixture of deionized 

water and n-butanol each at a ratio of 5:1 (mL/g), and vortexed at 1,000 rpm until it had been 

evenly distributed within the mixed solution. This solution was then centrifuged at 4,000 rpm for 

10  minutes,  to  produce  a  distinct  two-phase  system.  The  top  phase  consisted  of  a  mostly  clear 

butanol fraction, the bottom phase an opaquer green water fraction, with a white middle separating 

layer between the two. 

The butanol fraction was then collected via pipette, azeotroped with 5-10 mL of water and 

rotary evaporated under reduced pressure to leave a secondary green residue. This residue was 

collected from the flask with ethanol and introduced to a second centrifugation at 4,000 rpm for 

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10 minutes. After the second centrifugation, the precipitate formed at the bottom was collected 

and evaporated into a white powder that served as the saponin concentrate derived from soymeal.  

3.1.3 Quantification of Saponin in Derivation 

Analysis of saponins was performed by Liquid Chromatography/mass spectrometry (LC-

MS/MS)  using  a  Thermo  Q-Exactive  mass  spectrometer  interfaced  with  a  Thermo  Vanquish 

UHPLC (Thermo Fisher Scientific, MI, USA). Samples were either extracted with 80% methanol 

(solid  samples)  or  diluted  with  80%  methanol  (liquid  samples).  10  uL  of  sample  extract  was 

injected onto a Waters Acquity UPLC BEH-C18 column (2.1x100mm) (Waters Corp. MA, USA) 

and compounds were separated using the following gradient: initial conditions were 99% mobile 

phase A (10 mM ammonium formate in water) and 1% mobile phase B (acetonitrile + 0.1% formic 

acid) and held for 1 min followed by a ramp to 99% B at 16 min, held at 99% B until 18 min, then 

return to 1% B until 20 min. The flow rate was 0.3 ml/min and the column temperature was 40°C. 

Compounds were ionized by electrospray operating in positive ion mode with a capillary voltage 

of 3.5 kV and source gas and temperature settings adjusted for a 0.3 ml/min flow rate from the 

UHPLC. Data were acquired using a data-dependent MS/MS method with survey scans at 35,000 

resolution across m/z 100-1500, AGC target of 3e6 and max inject time of 100 ms. The top 5 ions 

were chosen for MS/MS scans at 17,500 resolution, AGC target of 1e5 and max inject time of 50 

ms, dynamic exclusion of 3 seconds, and stepped normalized collision energies of 10, 30 and 60 

V.  Raw  data  were  imported  into  Progenesis  QI  software  for  peak  alignment  and  peak  picking. 

Saponins peaks were identified based on accurate mass and fragmentation patterns. Quantities of 

all saponins were estimated using an external standard curve of soyasaponin I (Bb) obtained prior 

to the quantification of the samples. 

3.2 Renourishment of Soymeal 

The post-maceration soymeal attained after paper filtration was oven dried at 55 °C for at 

least 24 hours, and then the bottom fraction of the centrifuged solution mentioned in 3.1.2, was 

poured back onto it, allowing for nutrient reintegration via osmosis. 

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3.2.1 Verification of Nutrient Consistency after Reintroduction 

To verify that the bottom fraction reintroduction succeeded in replenishing the soymeal 

with the unnecessary extracted nutrients, a proximate composition analysis was performed. These 

composition  analyses  compared  the  5  major  components  (protein,  lipid,  mineral/ash,  moisture, 

carbohydrate) of soymeal samples both before extraction, and after the reintroduction. All soymeal 

samples tested were dried in a 55 °C oven for 24 hours prior to being analyzed and were done in 

at least 3 replications. 

3.2.1.1 Protein Content 

To determine the approximate protein content of the soymeal samples, a LECO FP828P 

Nitrogen  Analyzer  (LECO  Corp.  MI,  USA)  was  utilized.  Initially,  a  four-point  standard  curve 

(with  each  point  done  in  triplicate)  was  produced  with  different  quantities  of  nicotinic  acid. 

Thereafter, at least 3 blank samples were run to ensure proper calibration. Pre- and post-extraction 

soymeal samples between 0.2 and 0.3 g were then placed into the machine, where the N2 content 

was  analyzed.  To  translate  these  N2  values  into  protein  constituents,  a  Jones’  factor  of  6.25 

(industry standard) was utilized [4]. 

3.2.1.2 Mineral/Ash Content 

The values for the mineral/ash content of the soymeal samples were determined using a 

muffle furnace method in a Thermolyne B1 Muffle Furnace 208V (Thermo Scientific, MA, USA), 

following the protocol of [5]. Pre-weighed soymeal samples were set in porcelain crucibles and 

heated at 550 °C for 24 hours, cooled in a desiccator for 1-2 hours, and re-weighed against their 

initial weight to determine the mineral/ash content. 

3.2.1.3 Lipid Content  

The determination of the lipid/phospholipid content  of  the  soymeal  samples  was  carried 

out  through  the  use  of  a  microwave-assisted  (MA)  lipid  extraction  method  using  a  MARS6 

Microwave  Digestion  System  (CEM  Industries,  NC,  USA)  followed  by  gravimetric  analysis, 

following the protocol of [6]. The microwave extraction parameters involve a 1-stage, 400-Watt 

heating on a rotating carousel at 55 °C for 15 minutes.  

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0.5 g of each soymeal sample type was introduced to 8 mL of 4:1 ethyl acetate: methanol 

(+0.1% BHT as an antioxidant), put into pressurized 55 mm tubes and set into the carousel. The 

extract  was  then  transferred  to  clean  and  pre-weighed  10mL  glass  tubes  and  evaporated  under 

nitrogen  and  mild  heat  (37  °C).  These  conditions  were  used  to  prevent  the  oxidation  of  the 

extracted lipid product as much as possible. After evaporation, the lipid residue was weighed and 

divided against the initial 0.5 g sample to determine lipid content.   

3.2.1.4 Moisture Analysis 

Moisture values for all soymeal samples were determined using an A&D MX-50 Moisture 

Analyzer (A&D Company, MI, USA). Samples of 1g were weighed and placed into the moisture 

analyzer and heated until moisture content reached a rate of change of less than 0.1% /min. 

3.2.1.5 Carbohydrate Content 

The carbohydrate content of soymeal samples was determined using a difference method 

with the equation below, as is standard in proximate composition analyses of this nature [7]. 

% Carbohydrate = 100% - %Protein - %Lipid - %Mineral - %Moisture  

3.3 Preparation of Filler for Incorporation into Polymer Matrix 

3.3.1 Materials 

Crushed soybean hulls were provided by the Zeeland Farm Services soybean processing 

facility (Ithaca, MI, USA). These were cracked off the soybean via heating, they were then dried, 

and separated using air filtration during production.  

Sulfuric Acid (>96.4% purity) was obtained from J.T. Baker (Fischer Scientific, Hampton, 

NH, USA). Deionized water was obtained from the Michigan State School of Packaging. 

3.3.2 Particle Size Reduction of Soybean Hull Filler 

Soybean hulls were dried for 24 hours at 55 °C, milled for 15 minutes using an 8000D Dual 

Mixer/Mill (SPEX Sample Prep, Metuchen, NJ, 2010), and sieved through a 140 mesh (105 um) 

sieve  until  a  substantial  amount  of  material  was  attained  for  sample  production.  This  soyhull 

powder was stored in a Ziplock bag in a desiccator until film preparation.  

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3.3.3 Treatment of Powdered Soybean Hull Filler 

To provide a medium for saponins to interact with the filler particles, the procedure of [8] 

was  followed.  This  procedure  involved  the  immersion  of  filler  particles  into  a  water-saponin 

solution at a ratio of 10:90 v/v, followed by a 24-hour drying at 80 °C. The use of an acid catalyst 

of sulfuric acid (H2SO4) was investigated by inducing a pH of less than 2 during immersion. 

12 total samples (3 replicates for each treatment) of 2 g of soybean hull were immersed in 

26 mL of water in separate 200 mL beakers (with and without 0.05 g of saponin extract). 3 drops 

(0.15 mL) of sulfuric acid were then added to six of the samples (three with saponin and three 

without saponin) to provide the acid medium. All samples were then covered with foil and left at 

room  temperature  for  24  hours.  After  this,  the  samples  were  uncovered  until  the  liquid  had 

evaporated, and put into an oven at 55 °C for 24 hours to fully dry. After drying, the samples were 

crushed using a mortar and pestle to size with visibly large constituents removed and left to dry at 

55 °C for an additional 12 hours. The samples were then re-weighed and adjusted for consistency. 

3.4 Preparation/Fabrication of PLA/Soyhull Composites 

PLA pellets were pre-conditioned in an oven at 50 °C for 24 hours before being added to 

a  three-piece  mixer  (C.W  Brabender  Instruments  Inc.,  NJ)  rotating  at  80  rpm  at  180  °C  for  2 

minutes. Without stopping, the hull samples attained from Section 3.2.3 were gradually introduced 

into the mixer over the course of 30 seconds until all powder was incorporated, and melt-blended 

for an additional 4 minutes. Five different sample types were produced with 3 replicates of each, 

including a control of neat PLA. The composition and given titles of these composites are listed in 

Table 1.  

The composites were then pressed into sheets using a heated hydraulic press (PHI Model 

No. QL438-C, California, USA) between two 12” x12” x 0.6” Teflon coated stainless steel sheets 

(McMaster-Carr, IL, USA) at 183.9 °C under constant pressure of 7.5 tons for 2 minutes. Prior to 

the application of pressure, the resins were pre-heated on the hot press at the same temperature for 

6 minutes. After pressing, the composites were removed from the heat and left at room temperature 

for 15 minutes before being removed from the plates.  

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Table 1: Composition/title of PLA/Soyhull Composites. 

Title 

PLA 

Soyhull 

Saponin   

Neat PLA 

No Sap No H 

Yes Sap No H 

No Sap Yes H 

Yes Sap Yes H 

(g)  

19.8 

18 

18 

18 

18 

(g)  

0 

1.8 

1.8 

1.8 

1.8 

3.5 Characterization of Composites 

3.5.1 Mechanical Properties 

(g) 

0 

0 

0.045 

0 

0.045 

Total 

(g) 

19.8 

19.8 

19.845 

19.8 

19.845 

H2SO4 

Treatment 

N/A 

No 

No 

Yes 

Yes 

The  tensile  properties  of  all  composites  were  determined  using  an  Instron  Universal 

Testing Machine Model 5565 P6021 (Instron Engineering Corp. MA, USA). For preparation for 

tensile testing, samples were cut according to ASTM D882 [9] using a TMI Sample Cutter (RDM 

Test  Equipment  Ltd.,  UK)  and  measured  for  thickness  at  ten  separate  locations  using  a  digital 

micrometer (TMI). The samples used for tensile testing are shown in Figure 1. 

Samples were cut to 12.7 mm width and no less than 101.6 mm in length and tested with 

an initial grip separation of 50 mm and a rate of separation of 5.08 mm/min in accordance with 

ASTM D882 [9]. At least five samples were tested from each sheet produced. 

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37 

 
 
 
 
 
 
Figure 1: Image of the cut samples for tensile testing. 

3.5.2 Barrier Properties 

3.5.2.1 Oxygen Permeability 

The oxygen transmission rates of all composites were obtained using an Ox-Tran Model 

2/22 Oxygen Permeability Analyzer (Ametek, PA, USA) and then changed to oxygen permeability 

coefficients using the equation in [10]. Specimens from each treatment were cut from random sheet 

locations and masked with adhesive-backed 101.6um thick aluminum foil (McMaster Carr, IL, 

USA) with a testing area of 3.14 cm2. Samples were tested at 23 °C and 50% RH in accordance 

with ASTM D3985-17 [11]. Results are presented in kg*m/m2*Pa*day. At least two samples were 

tested from each sheet produced. 

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38 

 
 
 
 
 
 
 
 
 
3.5.2.2 Water Vapor Permeability 

The  water  vapor  transmission  rates  for  all  composites  were  determined  with  a  Mocon 

Permatran-W Model 3/34 Water Vapor Permeability Analyzer (Ametek, PA, USA). These were 

then translated to permeability coefficients using the equation in [10]. Specimens from each sheet 

were cut and masked with adhesive-backed 101.6 µm thick aluminum foil (McMaster Carr, IL, 

USA) with a testing area of 3.14 cm2. Samples were tested at 37.8 °C and 100% RH in accordance 

with ASTM F1249-20. [12]. Results are presented in kg*m/m2*Pa*day. At least two samples from 

each sheet produced were tested. 

3.5.3 Thermal Properties 

3.5.3.1 Thermal Gravimetric Analysis 

The  thermal  degradation  behavior  of  all  composites  was  determined  using  a  TGA  Q50 

Thermogravimetric analyzer (TA Instruments, Delaware, USA) with a nitrogen flow rate of 60mL 

per minute. 7-11 mg of sample was cut and put into a standard aluminum pan (TA Instruments, 

DE, USA) where it was subject to heating from 25 °C to 550 °C at a rate of 10 °C/minute. At least 

3 replications from each sheet produced was tested. 

3.5.3.2 Differential Scanning Calorimetry (DSC) 

The  crystallinity  and  thermal  properties  of  all  composites  were  determined  using 

differential scanning calorimetry DSC Q100 (TA Instruments, DE, USA) with a purge nitrogen 

gas flow rate of 70mL/min. 5-10mg of sample was cut and placed into a standard aluminum pan 

(TA Instruments, DE, USA) closed with a standard aluminum lid and then heated a rate of 10° 

C/min to 210 °C, down to -20 °C, and back up to 210 °C. The first heating was used to attain 

values  for  enthalpy  of  melting  (∆Hm),  and  enthalpy  of  cold  crystallization  (∆Hc),  the  second 

heating  was  used  for  the  determination  of  cold-crystallization  temperature  (Tc),  melting 

temperature  (Tm)  and  glass-transition  temperature  (Tg).  The  Universal  Analysis  2000,  V4.5 

software (TA Instruments, DE, USA) was used to determine the values Tc, Tm, Tg, ∆Hm, and ∆Hc. 

At least 3 replications from each sheet produced was tested. The equation used to calculate the 

percent crystallinity (%Xc) for PLA and the composites is below. 

%𝑋! = 	

 ∆Hm − ∆Hc
(1 − %𝑓𝑖𝑙𝑙𝑒𝑟) ∗ 93

∗ 100% 

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3.5.4 Morphological Analysis 

Images of the composites were attained using a scanning electron microscope (SEM) 

(JEOL 6610LV, JEOL, MA, USA). Samples were freeze-cracked using liquid nitrogen and 

coated with a thin layer of platinum prior to analysis. 

3.5.5 Optical Properties 

3.5.5.1 Color 

The color of the composite sheets was measured with a spectrophotometer (Labscan XE, 

Hunter Lab Associates Laboratory Inc., VA, USA) through a 2mm2 view area. After calibration, 

the CIELAB color coordinates L*, a*, and b* were attained from the EasyMatch QC software. 

Ten replications for each sheet produced was tested. 

3.5.5.2 Light Transmittance 

The light transmittance of each composite was determined with a UV/Vis 

spectrophotometer (Shimadzu UV 1800 Spectrophotometer Kyoto, Japan) and the UVProve 2.61 

software. Four replicate samples of equal thickness (0.32mm) from each sheet were cut and 

placed into the machine. The absorbance was taken from 200 nm to 800 nm with measurements 

taken every 0.5nm at “medium” speed. The absorbance spectra are displayed in nm vs. 

absorbance for the full spectrum measured.  

3.6 Statistical Analysis 

To perform the statistical analysis for the tensile, barrier, and color data, a two-way 

ANOVA test was performed with a p-value of 0.05. To perform the statistical analyses for DSC 

results, a linear mixed model was incorporated using the package ‘ImerTest' in the R program. 

Mean separation and letter differentiation was performed using the ‘emmeans’ package in R. 

Post hoc comparisons of the data was conducted using Tukey’s adjustment, and a significance 

value of p < 0.05. The alpha level for all analyses was 0.05, and the p-value for multiple 

comparisons was corrected by Bonferroni correction. 

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REFERENCES 

Hu, J., Lee, S.-O., Hendrich, S., & Murphy, P. A. (2002). Quantification of the group B 

1. 
soyasaponins by high-performance liquid chromatography. Journal of Agricultural and Food 
Chemistry, 50(9), pp 2587–2594. https://doi.org/10.1021/jf0114740 

Decroos, K., Vincken, J.-P., van Koningsveld, G. A., Gruppen, H., & Verstraete, W. 

2. 
(2007). Preparative chromatographic purification and surfactant properties of individual 
soyasaponins from soy hypocotyls. Food Chemistry, 101(1), pp 324–333. 
https://doi.org/10.1016/j.foodchem.2006.01.041 

3. 
Shimoyamada, M., Okubo, K., Yoshikoshi, M., Watanabe, K., ,. (1995). Partition of 
soybean saponins between butanol and water. Food Science and Technology International, 
Tokyo, 1(2), pp 112–114. https://doi.org/10.3136/fsti9596t9798.1.112 

4. 
Mariotti, F., Tomé, D., & Mirand, P. P. (2008). Converting nitrogen into protein—
beyond 6.25 and jones’ factors. Critical Reviews in Food Science and Nutrition, 48(2), pp 177–
184. https://doi.org/10.1080/10408390701279749 

Harris, G. K., & Marshall, M. R. (2017). Ash analysis. Food Science Text Series, 287–

5. 
297. https://doi.org/10.1007/978-3-319-45776-5_16 

6. 
AOAC - Official Method 985.15. Fat (crude) in meat and poultry products. Rapid 
microwave-solvent extraction method Official Methods of Analysis of AOAC International (19th 
ed.), AOAC International, Gaithersburg, MD, USA (2012) 

University of Massachussetts. (n.d.). Analysis of Carbohydrates. 7. analysis of 

7. 
carbohydrates. (2015) 

Cherian, B. M., Leão, A. L., Caldeira, M. da, Chiarelli, D., de Souza, S. F., Narine, S., & 

8. 
de Morais Chaves, M. R. (2012). Use of Saponins as an effective surface modifier in cellulose 
nanocomposites. Molecular Crystals and Liquid Crystals, 556(1), pp 233–245. 
https://doi.org/10.1080/15421406.2012.635969 

9. 
American Society for Testing and Materials: “ASTM D0882-18 Standard Test Method 
for Tensile Properties of Thin Plastic Sheeting”, American Society for Testing and Materials, 
April 2018 

Pranata, M. P., González-Buesa, J., Chopra, S., Kim, K., Pietri, Y., Ng, P. K., Matuana, 

10. 
L. M., & Almenar, E. (2019). Egg white protein film production through extrusion and 
calendering processes and its suitability for food packaging applications. Food and Bioprocess 
Technology, 12(4), pp 714–727. https://doi.org/10.1007/s11947-019-2248-0 

American Society for Testing and Materials: “ASTM D3985-18: Standard Test Method 

11. 
for Oxygen Gas Transmission Rate Through Plastic Film and Sheeting Using a Coulometric 
Sensor” American Society for Testing and Materials (2018) 

American Society for Testing and Materials: “ASTM F1249-20 Standard Test Method 

12. 
for Water Vapor Transmission Rate Through Plastic Film and Sheeting Using a Modulated 
Infrared Sensor American Society for Testing and Materials, April 2020 

41 
41 

 
 
 
 
 
 
 
 
CHAPTER 4 

RESULTS & DISCUSSION 

4.1 Quantification of Soyasaponin Extract 

The white precipitate remaining after centrifugation in 3.1.2 was dried and estimated to 

consist of  >51% soyasaponin (>33% soyasaponin Bb (I), specifically). The remaining components 

were  deemed  to  be  largely  isoflavones  (genistein,  daidzein,  and  glycitein),  with  a  small 

lipid/phospholipid portion based on the HPLC/MS results. This purity was slightly lower than [1] 

and [2], that performed similar procedures for saponin extraction. Possible reasons for this include 

the derivation source, polarity of the maceration solvent, and singular partitioning. 

Soy isoflavones, the other major component of the extract, are distinguished in structure as 

well and contain ample reactive sites to interact with the filler particles and potentially alter their 

surface chemistry [3]. 

Due  to  this  purity  level,  and  the  chemical  composition  of  the  remaining  fraction  of  the 

extract, the properties attributed to this extract cannot be isolated to soyasaponins alone. 

4.2 Soymeal Nutrient Evaluation 

4.2.1 Proximate Composition Analysis of Soymeal After Extraction & Reintroduction 

Table 2: Proximate composition analysis of the soybean meal before the extraction and 

after the reintroduction process. 

Content (%) 

Constituent 

Original soymeal 

Improved soymeal 

Protein 

Lipid 

Mineral/Ash 

Moisture 

Carbohydrate (make-up 

compound) 

Saponin 1. 

51.60 ± 0.69a 

2.91 ± 0.97a 

6.28 ± 0.10a 

1.55 ± 0.21a 

37.66a 

52.09 ± 1.42a 

0.36 ± 0.00b 

6.37 ± 0.09a 

2.95 ± 0.00b 

38.23a 

1.25 ± 0.12a 

0.31 ± 0.00b 

Different lower-case letters (a, b) indicate significant differences between the columns. 

42 
42 

 
 
 
 
 
 
 
 
 Table 2 (cont’d) 
 1. Saponins within the meal are considered a subcategory of the lipid and carbohydrate content 
and thus are already included in the overall composition value [4, 5]. 

The values shown in Table 2 for the original meal are consistent with USDA standards 

for defatted soybean meal [6].  

4.2.1.1 Protein Content 

The protein content of the post-extraction meal was consistent with that of the original 

meal sample. The use of a less polar solvent in 60% ethanol, and the maceration temperature of 

23 °C was incorporated to limit protein solubilization and prevent denaturization [7]. Due to 

protein content being the largest source of appeal for defatted soymeal, the retention of this 

content is extremely important for the value of the post-extraction meal. 

4.2.1.2 Mineral/Ash Content 

The mineral/ash content within the meal also remained consistent after extraction. This 

was mostly expected as most minerals present in soymeal are not as readily soluble in water or 

other polar solvents [8]. 

4.2.1.3 Lipid Content 

The lipid composition of the meal after extraction and reintroduction procedures was 

notably lower (<15% of original value) than the original meal. A loss of value due to this 

difference in lipid content could be considered as lipids provide essential energy storage and 

absorption for the livestock consuming them [9]. However, due to the notion that the meal is 

already defatted, the initial lipid content is extremely low, and livestock are typically 

supplemented with their dietary lipids from other food sources regardless [10]. 

4.2.1.4 Moisture Content 

The moisture content of the meal is higher after the extraction process in comparison to 

the initial meal. This moisture content likely higher due to the primary and secondary 

immersions in water/ethanol the post-extraction meal undergoes during the maceration, and 

reintroduction phases. This integrates the water further into the microstructure of the meal, 

making it more difficult and time consuming to evaporate. Since the drying procedure for these 

43 
43 

 
 
 
 
 
 
 
samples was only at 55 °C for 24 hours, it is reasonable to assume that this additional water may 

not have had enough energy to leave. 

4.2.1.5 Carbohydrate Content 

The broad carbohydrate content of the meal is undifferentiable from the original soymeal. 

This is vital in the continuation of the post-extraction soymeal as an energy-rich animal feed. 

4.2.2 Saponin Content 

The saponin content in the initial meal was slightly higher than other values seen from 

defatted soymeal [5, 11]. This may be due to the growing conditions of the soybean effecting 

these values, or the type of defatting process the soymeal was subject to compared to others [12]. 

Saponin content of the post-maceration meal is less than a third than that of the initial. This 

proves the extraction mechanism was successful in separating a majority of the saponins existing 

in the meal at least in the butanol fraction. Macerating in a less polar solvent, or repeated 

partitioning may increase this extraction effectiveness. Regardless, a meal source with less 

saponins in it is expected to be less harmful to the health and production of livestock such as 

cattle and fish. 

44 
44 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.3 Characterization of PLA-Soybean Hull Composites 

4.3.1 Mechanical Properties  

Figure 2: Tensile strength, elongation at break, and elastic moduli of all composites. 

45 
45 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Table 3: Tensile test results for all composites. 
Tensile Strength 

Sample 

Elongation at Break (%) 

(MPa) 

MOE 

(MPa) 

Neat PLA 

50.41 ± 3.60 a 

4.14 ± 0.55 a 

1909.64 ± 120.69 a 

No Sap No H 

38.00 ± 2.18 b 

3.36 ± 0.41 bc 

2023.64 ± 108.87 ab 

Yes Sap No H 

40.35 ± 1.84 c 

No Sap Yes H 

37.58 ± 2.33 b 

Yes Sap Yes H 

40.29 ± 2.17 c 

3.38 ± 0.30 b 

3.05 ± 0.24 c 

3.50 ± 0.36 b 

2161.54 ± 115.00 b 

1980.54 ± 175.40 ab 

2370.49 ± 354.49 b 

Different lowercase letters (a, b, c) indicate groupings that are statistically different from 

another.  

The inclusion of the soyhull filler decreased the strength of the PLA composites. This is 

indicated by the lower tensile strength values for composites with all filler treatments compared 

to the neat PLA. This loss in strength was expected due to the inclusion of a mostly incompatible 

substance into the plastic matrix [13]. 

The treatment of the filler with the saponin extract improved the composites tensile 

strength in comparison to the non-saponin treated samples, however, this improvement was still 

considerably less than the values for the neat PLA. The acid treatment of the filler prior to 

inclusion did not affect the tensile strength of composites in comparison to the non-acid treated 

samples. The combination of both the saponin and acid treatments resulted in a composite with 

improved strength compared to the untreated and solely acid treated samples, however this level 

of improvement was not considered different from the solely saponin-treated filler. This 

indicates that the combination of the two treatments did not initiate any additional effect between 

the saponin and the filler in this area.   

The inclusion of the soyhull filler also decreased the elongation values of composites 

regardless of the treatment in comparison to the neat PLA as expected [13]. All treatment 

variations of saponin, acid, and the combination of the two provided no improvements in this 

area compared to composites produced from the untreated filler. This limits the belief of a 

saponin-induced improvement, or that there is a significant catalysis in saponin interaction from 

the acid medium. 

46 
46 

 
 
 
 
 
 
 
The elastic modulus of composites did not change with the inclusion of the soyhull filler. 

An increase in this area was expected due to the rule of mixture [14], however the loading factor 

of 10% may have limited the extent of this effect. The use of the saponin-treated filler resulted in 

a modulus significantly higher than the neat PLA, however, it was indifferentiable from the 

untreated filler composites. The acid treatment of the filler shows no difference in comparison to 

both the neat plastic and the untreated filler. The combination of the saponin and acid treatments 

on the filler produced composites with higher moduli than that of the neat PLA but was still 

indifferentiable from the untreated filler composites.  

4.3.2 Barrier Properties 

Table 4: Oxygen Permeability (OP) and Water Vapor Permeability (WVP) for all 

composites. 

Sample 

OP 

WVP 

* E-18 (kg*m/m2*Pa*s) 

 * E-14 (kg*m/m2*Pa*s) 

Neat PLA 

No Sap No H 

Yes Sap No H 

No Sap Yes H 

Yes Sap Yes H 

4.24 ± 0.29 a 

4.00 ± 0.39 a 

3.96 ± 0.40 a 

4.17 ± 0.26 a 

3.87 ± 0.42 a 

1.85 ± 0.03 a 

1.93 ± 0.04 b 

1.92 ± 0.05 b 

1.88 ± 0.04 ab 

1.84 ± 0.04 a 

Different lowercase letters (a, b) indicate groupings that are statistically significant from 

another. 

47 
47 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.3.2.1 Oxygen Permeability 

Figure 3: Oxygen permeability coefficients for all composites in kg*m/m2*Pa*s. 

The inclusion of the filler regardless of the treatment had no effect on the oxygen 

permeability of the composites leaving comparable values to the neat PLA. This is likely 

attributed to the tortuosity effect that fillers have been known to induce for composites [15]. This 

provides a more tortuous path with more obstacles for permeants, particularly less polar ones 

such as oxygen. This effect in turn mitigates the otherwise negative effect fillers can have on the 

uniformity of the polymer structure on permeability. 

48 
48 

 
 
 
 
 
 
 
 
 
 
 
4.3.2.2 Water Vapor Permeability 

Figure 4: Water vapor permeability values for all composites in kg*m/m2*Pa*s. 

The inclusion of the untreated soyhull showed statistically significant reductions in the 

material’s barrier to water, the resultant permeability was more than 5% higher than the neat 

PLA. This is often seen in fillers of similar nature due to their hydrophilicity [13, 16]. The 

saponin treatment of the filler did not have a significant effect on the WVP of composites when 

compared to the untreated composites, retaining a significant deterioration in this category 

compared to the neat plastic. The acid treatment of the filler showed no effect on WVP values in 

comparison to the untreated filler-composites, yet the acid treated composites were also 

indistinguishable from neat PLA. The combination of the saponin and acid treatments resulted in 

a water vapor barrier significantly better than both non-acid treated composites and comparable 

to the neat PLA. Improvements in this area are often seen from from both acid-treated fillers and 

from coupling agents because of the modified surface chemistry involved with them [13, 16]. 

49 
49 

 
 
 
 
 
 
 
These improvements are generally attributed towards the tortuosity effect mentioned earlier, as 

well as a decreased hydrophilicity of the filler after treatment.  

4.3.3 Thermal Properties 

4.3.3.1 Thermogravimetric Analysis 

Figure 5: Displays the weight change (%) as a function of Temperature (° C) observed 

during Thermogravimetric Analysis for all composites.  

There is an initial loss observed for the filler-composites around 100-120 °C. This loss is 

less than 1% of the total weight and is attributed to the loss of water that may have remained in 

the samples [17]. All composites showed a further increase in weight loss of an additional 1-2% 

between 210-290 °C that was not observed in the neat PLA. This secondary loss can be 

indicative of the initial loss of hemicellulose and lignin [18, 19]. The third step in weight loss is 

observed between 300 and 370 °C, where over 90% of the weight was lost. This is widely 

accepted as the degradation temperature range of PLA, and is also a result of the decomposition 

of cellulose in the filler-composites [20].  

The weight loss was maximized at 350-355 °C for the non-acid treated composites, and at 

380-385 °C for the acid treated and neat resins. The non-acid treated hulls contain large 

50 
50 

 
 
 
 
 
 
 
quantities of cellulose which decompose at 270-330 °C [21], this may have initiated the weight 

loss at an earlier stage [22]. The hydrolysis of the cellulose in the soyhulls induced from the acid 

treatment likely produced higher contents of oligosaccharides in place of cellulose, which have 

higher degradation temperatures, thus diminishing this earlier onset degradation [19, 22]. There 

is no observed effect of the saponin-treatment on thermal stability of the composites.  

4.3.3.2 Differential Scanning Calorimetry (DSC) 

Table 5: Glass transition temperature (Tg), cold crystallization temperature (Tc), melting 

temperature (Tm), and the percent crystallinity (Xc) of all composites. 

Sample 

Neat PLA 

No Sap No H 

Yes Sap No H 

No Sap Yes H 

Yes Sap Yes H 

Tg (°C)  Tc (°C) 

Tm (°C) 

Xc (%) 

60.81a 

112.91a 

148.12a 

60.33a 

112.65a 

147.70a 

60.74a 

112.94a 

148.43a 

59.56a 

111.22b 

146.98b 

59.47a 

112.25ab 

146.92b 

15.54a 

20.69b 

18.92b 

19.49b 

19.52b 

Different lowercase letters (a, b) in each column indicate groupings that are statistically 

significant from another. 

The inclusion of the soyhull filler showed no effect on the Tg of composites regardless of 

the treatment in comparison to neat PLA. The filler loading is not considered high enough to 

have a drastic effect on the transition temperature [23].  

The untreated, and saponin-treated soyhull composites showed no difference in Tc in 

comparison to the neat control. However, the acid treatment of the filler showed a markable 

decrease in this value. This may be attributed to the non-washing of the acid residue off the filler 

prior to inclusion. Sulfuric acid has been cited in previous literature to catalyze transesterification 

reactions between the esters in the PLA mainchain during extrusion. This typically leads to 

shorter chain lengths, less overlapping, and thus less required energy for movement among them 

[24, 25]. The combination of both acid and saponin treatments had no effect on this value 

compared to all other samples produced.  

The inclusion of the non-acid treated filler showed little effect on the Tm of composites 

compared to the neat PLA. The acid treatment of the filler showed a decrease in this value. This 

can also be attributed to the potential transesterification reaction the residual H2SO4 may have 

51 
51 

 
 
 
 
 
 
 
catalyzed during extrusion, thus reducing the energy required for freedom of movement of the 

chains. The combination of the saponin and acid treatments showed a similar melting 

temperature to the acid-treated sample, statistically lower than the other treatment variations. 

The presence of the filler increased the crystallinity content of the PLA; however, this 

increase was not greatened by either saponins or acidification. The difference of percent 

crystallinity with the inclusion of all treatment variations of soybean hulls indicates their 

viability as a nucleating agent [26, 27]. The incorporation of other coupling agents has shown 

improvements in Xc, this is generally attributed to the improved adhesion into the polymer, 

allowing for more uniformity within the matrix [28, 29].  

Figure 6: Heat Flow in (W/g) as a function of temperature from –20 to 215 °C for all 
composites. The Tg, Tc, and Tm temperature values for the neat PLA (bottom) are exemplified 
with vertical lines for comparison.  

The establishment of a double peak in the melting temperature range for the 10% SH 

composites has been noted frequently in PLA composites including organic fillers [27, 28, 30]. 

This double peak is generally attributed to a double lamellar thickness within the composite 

structure. Specifically, there may be two separate crystalline phases comprised of different 

“perfection” levels that thereby melt at different temperatures. It is generally assumed that 

52 
52 

 
 
 
 
 
 
          
 
 
whichever structure is less “perfect” is responsible for the earlier peak, where the more organized 

lattice is melted at a higher temperature [27, 28]. The presence of this second peak appears to 

diminish with the presence of saponins under both acid and non-acid treated conditions. This 

may be evidence of the saponins ability to provide a more uniform crystalline structure in 

composites. 

4.3.4 Morphological Analysis 

Figure 7: Displays the SEM microscopic images for all composites; a.). Neat PLA, b.) 

No Sap No H, c.) Yes Sap No H, d.) No Sap Yes H, and e.) Yes Sap Yes H. 

Images are shown at both 250x (1st and 2nd row), and 500x (3rd row) magnitudes for visual 
comparison. 

The images above display an estimation for the extent of adhesion for some of the filler 

particles given the different treatments. The presence of visible gaps between the filler particles 

and the matrix indicates a poor interfacial adhesion between the two, this is seen clearly in the 

500x magnitude image for b.). The images in c.) provide excellent visuals for a well adhered 

filler into the matrix, this adhesion can perhaps explain the better mechanical properties observed 

in the fillers from the saponin treatment. 

53 
53 

 
 
 
 
 
 
 
 
 
 
 
4.3.5 Optical Characterization 

4.3.5.1 Color 

Table 6: Lightness (L*), and color coordinates a* and b* of the composite and neat PLA 

sheets. 

Sample 

L* 

a* 

b* 

Neat PLA 

89.85 ± 0.26a 

-1.27 ± 0.00a 

4.57 ± 0.21a 

No Sap No H 

77.33 ± 1.41b 

2.33 ± 0.43b 

22.25 ± 1.56b 

Yes Sap No H 

76.412 ± 1.48b 

2.80 ± 0.49b 

24.27 ± 1.53bc 

No Sap Yes H 

65.70 ± 5.14c 

7.21 ± 1.42c 

23.99 ± 2.16c 

Yes Sap Yes H 

64.74 ± 2.99c 

8.38 ± 0.90d 

26.39 ± 1.17d 

Different lowercase letters (a, b) in each column indicate groupings that are statistically 

significant from another. 

The neat PLA shows L*, a*, and b* values resembling a grayish clear color, with a slight 

greenish-yellow tint, as expected. The addition of the soyhulls to the PLA resulted in 

significantly darker composites. This is seen often with filler-based composites due to differing 

light-reflection properties [31]. The inclusion of the soyhulls into the matrix produced 

significantly higher values on both the b* spectrum and a* spectrum. The inclusion produced 

sheets that were more red and yellow in appearance. The acid treatment of the filler prior to 

incorporation further increased this redness and yellowness in the resultant composite. This color 

change is likely due to the alteration of the surface chemistry of the fillers, thus disrupting the 

usual wavelength reflectiveness range [32]. 

The saponin treatment had a significant effect on the a* and b* values. Although it only 

comprised 0.25 wt.% of the entire composite, simply the presence of these compounds may have 

worked to alter these values. The saponin extract itself had a yellowish tint to it, providing reason 

for the higher b* values observed. 

54 
54 

 
 
 
 
 
 
 
 
 
 
 
4.3.5.2 Light Transmittance 

Figure 8: Transmittance of the composite sheets and neat PLA sheets  through the 

visible, UVA, UVB, and some of UVC light spectrum. 

As expected, the inclusion of a filler at a 10% loading reduced the transparency of the 

film through visible light by nearly 70%. This is largely attributed to the different structures 

within the soybean hull fillers, absorbing a vast majority of the light in the visible and UV 

spectrum when compared to the neat plastic. This finding is consistent with others in similar 

studies [31]. Although the decreased transmittance diminishes visible appeal of the composites, 

the continued absorbance of light in the UV spectrum bodes well for the material as a UV-

blocker in light-sensitive food products.  

55 
55 

 
 
 
 
 
 
 
 
 
REFERENCES 

1. 
Knudsen, D., Urán, P., Arnous, A., Koppe, W., & Frøkiær, H. (2007). Saponin-
containing subfractions of soybean molasses induce enteritis in the distal intestine of Atlantic 
salmon. Journal of Agricultural and Food Chemistry, 55(6), 2261–2267. 
https://doi.org/10.1021/jf0626967  

2. 
Shimoyamada, M., Okubo, K., Yoshikoshi, M., Watanabe, K., ,. (1995). Partition of 
soybean saponins between butanol and water. Food Science and Technology International, 
Tokyo, 1(2), 112–114. https://doi.org/10.3136/fsti9596t9798.1.112  

Barnes, S. (2010). The biochemistry, chemistry and physiology of the isoflavones in 

3. 
soybeans and their food products. Lymphatic Research and Biology, 8(1), 89–98. 
https://doi.org/10.1089/lrb.2009.0030  

Shi, J., Arunasalam, K., Yeung, D., Kakuda, Y., Mittal, G., & Jiang, Y. (2004). Saponins 

4. 
from edible legumes: Chemistry, processing, and Health Benefits. Journal of Medicinal Food, 
7(1), 67–78. https://doi.org/10.1089/109662004322984734  

Knudsen, D., Røn, Ø., Baardsen, G., Smedsgaard, J., Koppe, W., & Hanne Frøkiær. 

5. 
(2006). Soyasaponins resist extrusion cooking and are not degraded during gut passage in 
Atlantic Salmon (salmo salar L.). Journal of Agricultural and Food Chemistry, 54(17), 6428–
6435. https://doi.org/10.1021/jf0604992  

Tsai, A. M., van Zanten, J. H., & Betenbaugh, M. J. (1998). I. Study of protein 

6. 
aggregation due to heat denaturation: A structural approach using circular dichroism 
spectroscopy, nuclear magnetic resonance, and static light scattering. Biotechnology and 
Bioengineering, 59(3), 273–280. https://doi.org/10.1002/(sici)1097-
0290(19980805)59:3&lt;273::aid-bit2&gt;3.0.co;2-8  

Bernard, J. K, (2022) Oilseed and Oilseed Meals, Encyclopedia of Dairy Sciences (Third 

7. 
Edition) pp 614-619 doi: 10.1016/B978-0-12-818766-1.00103-3 

8. 

University of Utah. (2007). Mineral Decomposition. Chapter 6: Weathering and Soil 

Kerr, B. J., Kellner, T. A., & Shurson, G. C. (2015). Characteristics of lipids and their 

9. 
feeding value in swine diets. Journal of Animal Science and Biotechnology, 6(1). 
https://doi.org/10.1186/s40104-015-0028-x  

Riaz, M. N., & Alam, M. S. (2020). Fats and oils in feedstuffs and Pet Foods. Bailey’s 

10. 
Industrial Oil and Fat Products, 1–66. https://doi.org/10.1002/047167849x.bio074.pub2  

11. 
Ireland, P. A., Dziedzic, S. Z., & Kearsley, M. W. (1986). Saponin content of soya and 
some commercial soya products by means of high‐performance liquid chromatography of the 
sapogenins. Journal of the Science of Food and Agriculture, 37(7), 694–698. 
https://doi.org/10.1002/jsfa.2740370715  

12.  MacDonald, R. S., Guo, J., Copeland, J., Browning, J. D., Sleper, D., Rottinghaus, G. E., 
& Berhow, M. A. (2005). Environmental influences on isoflavones and saponins in soybeans and 
their role in colon cancer. The Journal of Nutrition, 135(5), 1239–1242. 
https://doi.org/10.1093/jn/135.5.1239  

56 
56 

 
 
 
 
 
 
13. 
H.T. Duguma, P. Khule, A. McArdle, K. Fennell, E. Almenar, Turning agricultural waste 
into packages for food: A literature review from origin to end-of-life, Food Packaging and Shelf 
Life. 40 (2023) 101166. doi:10.1016/j.fpsl.2023.101166.  

Srinivasababu, N., (2019) 6 – Edge crack effect on tensile behavior of diversified wood 

14. 
particulate composites, Filer Analysis in Biocomposites Fibre-Reinforced Composites and 
Hybrid Composites, Woodhead Publishing Series in Composites Science and Engineering, pp 
109-131 doi: 10.1016/B978-0-08-102293-1.00006-1 

Idris, A., Muntean, A., Mesic, B., (2022) A review on predictive tortuosity models for 

15. 
composites films in gas barrier applications, Journal of Coatings Technology and Research, 
Chapter 19 pp 699-716 doi: 10.1007/s11998-021-00579-6   

16.  Wolf, C.,  Angellier-Coussy, H., Gontard, N., Doghieri, F., Guillard, V., (2018) How the 
shape of filler affects the barrier properties of polymer/non-porous particle nanocomposites: A 
review, Journal of Membrane Science Vol. 556 pp 393-418 doi: 10.1016/j.memsci.2018.03.085  

17. 
Zapata, B., Balmaseda, J., Fregoso-Israel, E., & Torres-García, E. (2009). Thermo-
Kinetics Study of Orange Peel in Air. Journal of Thermal Analysis and Calorimetry, 98(1), 309–
315. https://doi.org/10.1007/s10973-009-0146-9  

18. 
Yamoum, C., & Magaraphan, R. (2015). Effect of peanut shell content on mechanical, 
thermal, and biodegradable properties of peanut shell/polylactic acid biocomposites. Polymer 
Composites, 38(4), 682–690. https://doi.org/10.1002/pc.23627  

Yang, H., Yan, R., Chen, H., Lee, D. H., & Zheng, C. (2007). Characteristics of 

19. 
hemicellulose, cellulose and lignin pyrolysis. Fuel, 86(12–13), 1781–1788. 
https://doi.org/10.1016/j.fuel.2006.12.013  

20. 
Fan, Y., Nishida, H., Shirai, Y., Tokiwa, Y., & Endo, T. (2004). Thermal degradation 
behaviour of poly(lactic acid) Stereocomplex. Polymer Degradation and Stability, 86(2), 197–
208. https://doi.org/10.1016/j.polymdegradstab.2004.03.001  

D’Acierno, F., Michal, C. A., & MacLachlan, M. J. (2023). Thermal stability of cellulose 

21. 
nanomaterials. Chemical Reviews, 123(11), 7295–7325. 
https://doi.org/10.1021/acs.chemrev.2c00816  

Kowalczyk, M., Piorkowska, E., Kulpinski, P., & Pracella, M. (2011). Mechanical and 

22. 
thermal properties of PLA composites with cellulose nanofibers and standard size fibers. 
Composites Part A: Applied Science and Manufacturing, 42(10), 1509–1514. 
https://doi.org/10.1016/j.compositesa.2011.07.003  

23. 
Droste, D. H., Dibenedetto, A. T., (1969) The g;ass transition temperature of filler 
polymers and its effect on their physical properties Journal of Applied Polymer Science, Vol. 13 
Issue 10 pp 2149-2168 doi: 10.1002/app.1969.070131011 

Suchaiya, V., & Aht-Ong, D. (2014). Microwave-assisted modification of cellulose as a 

24. 
compatibilizer for PLA and MCC biocomposite film: Effects of side chain length and content on 
mechanical and thermal properties. Polymers and Polymer Composites, 22(7), 613–624. 
https://doi.org/10.1177/096739111402200705  

57 
57 

 
 
 
 
 
 
Elsawy, M. A., Kim, K.-H., Park, J.-W., & Deep, A. (2017). Hydrolytic degradation of 

25. 
polylactic acid (PLA) and its composites. Renewable and Sustainable Energy Reviews, 79, 
1346–1352. https://doi.org/10.1016/j.rser.2017.05.143  

Xia, X., Shi, X., Liu, W., Zhao, H., Li, H., & Zhang, Y. (2017). Effect of flax fiber 

26. 
content on polylactic acid (PLA) crystallization in PLA/flax fiber composites. Iranian Polymer 
Journal, 26(9), 693–702. https://doi.org/10.1007/s13726-017-0554-9  

27. 
Shieh, Y., & Liu, G. (2007). Effects of carbon nanotubes on crystallization and melting 
behavior of poly(L‐lactide) via DSC and TMDSC studies. Journal of Polymer Science Part B: 
Polymer Physics, 45(14), 1870–1881. https://doi.org/10.1002/polb.21184  

Pilla, S., Gong, S., O’Neill, E., Yang, L., & Rowell, R. M. (2008). Polylactide‐Recycled 

28. 
Wood Fiber Composites. Journal of Applied Polymer Science, 111(1), 37–47. 
https://doi.org/10.1002/app.28860  

Anbupalani, M. S., Venkatachalam, C. D., & Rathanasamy, R. (2020). Influence of 

29. 
coupling agent on altering the reinforcing efficiency of natural fibre-incorporated polymers – A 
Review. Journal of Reinforced Plastics and Composites, 39(13–14), 520–544. 
https://doi.org/10.1177/0731684420918937  

Tisserat, B., Joshee, N., Mahapatra, A. K., Selling, G. W., & Finkenstadt, V. L. (2013). 

30. 
Physical and mechanical properties of extruded poly(lactic acid)-based paulownia elongata 
biocomposites. Industrial Crops and Products, 44, 88–96. 
https://doi.org/10.1016/j.indcrop.2012.10.030  

31. 
Fehlberg, J., McKay, S., Matuana, L. M., & Almenar, E. (2023). Use of orange juice 
processing waste to produce films using blown film extrusion for food packaging. Journal of 
Food Engineering, 341, 111337. https://doi.org/10.1016/j.jfoodeng.2022.111337  

SÖĞÜT, E., & SEYDİM, A. C. (2022). Utilization of kiwi peel lignocellulose as fillers in 

32. 
poly(lactic acid) films. Journal of the Turkish Chemical Society Section A: Chemistry, 9(1), 
283–294. https://doi.org/10.18596/jotcsa.1024326  

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CHAPTER 5  

CONCLUSION 

5.1 Conclusion 

Soyasaponins were able to be safely and effectively extracted from defatted soymeal 

without harming significant macromolecules of the meal including proteins, minerals, and 

carbohydrates. The treatment of the soyhull filler with the saponin extract prior to extrusion was 

able to produce PLA-soyhull composites with improved tensile strength compared to the 

untreated composites, providing evidence of viability as a coupling agent. The saponin treatment 

also provided evidence for more uniformity in composite crystallinity from the DSC curve 

results.  

The acid treatment of the soyhulls prior to extrusion produced composites with improved 

water barrier, thermal degradation resistance, and UV barrier properties in comparison to the 

untreated composites. However, this treatment also coincided with lower Tc, and Tm values, the 

non-washing of the acid residue on the filler is believed to have played a significant role in these 

observations. 

The combination of the acid and saponin treatments produced a composite with 

significant improvements in tensile strength in comparison to the untreated composites. 

However, these improvements were not notably greater than the composites including the 

saponin treatment alone. This treatment combination also showed improved WVP, thermal 

resistance, and UV barrier properties in comparison to the untreated filler composites. It also 

produced composites with decreased Tc and Tm, limiting the potential for some food packaging 

applications. 

The proof of a safe extraction method of soyasaponins from soymeal and the 

establishment of their non-necessity in meal for some livestock provides a fully sustainable 

source for soyasaponins with commercial appeal for their extraction. 

The potential of soyasaponins as an agent to improve tensile properties breaks significant 

ground in what was otherwise a minimally investigated field. The parameters used while testing 

including the concentration, mechanism of action, and sample production are all areas where 

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improvement can be made. This work provides a basis for others in discovering other naturally 

occurring coupling agents, testing saponins from other sources, and the overall betterment of 

green plastic composites.  

The effect on acidification is not consistent with most literature in improving mechanical 

properties. However, the non-washing of the fillers after treatment gives difficulty in providing a 

true assessment. The acid treatment did not appear to hinder or improve the effectiveness of the 

saponin treatment. 

5.2 Future Work 

The effectiveness of the post-extraction soymeal as an improved food source for cattle 

and fish should be investigated for confirmation.  

The evaluation of differing concentrations of the soyasaponin relative to the waste would 

be significant in attaining an optimal effectiveness. The investigation into evidence of the 

chemical reactions taking place as a result of the saponin treatment would be beneficial for fuller 

comprehension. Usage of the extract with fillers and plastics other than soyhull and PLA can also 

be investigated. The effectiveness of the saponin extract in comparison to commercially 

available coupling agents would provide an idea for the level of improvement. A look into the 

potential antimicrobial and antioxidant inducing properties of saponins on composites could be 

promising. Finally, the effect on the resultant biodegradability of composites after the saponin 

extract inclusion is necessary to prove the full viability of a truly green coupling agent. 

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