testing for starch experiment

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• Test For Starch

Test for Starch

What is starch.

In simple words, starch can be defined as the most important complex carbohydrate compounds. It is a polysaccharide and glucoside reserve of plants. It is a renewable and biodegradable product, so it can act as a perfect raw material and a substitute for fossil-fuel components in making detergents, glues, plastics, etc.

The starch molecules comprise a large number of glucose units that are bound together by glycosidic bonds and are produced by all vegetables and other plant sources through the process of photosynthesis . The starch molecules function as energy storage in plant cells, which is necessary for their growth, development, and reproduction. Barley, potatoes, maize, rice, wheat are a few examples of plant products from which starch are extracted and distributed to different industries.

Also Refer:  Carbohydrates

Why do we perform Test for Starch?

The iodine test for starch is mainly performed to test the presence of carbohydrates. The food products which we eat include different types of carbohydrates, among which starch and sugars are the main carbohydrates found in our food products.

Also Read:  Sources of Carbohydrates

Test for Starch in Plants Source

Materials required.

• Porcelain tile.
• Iodine solution
• Food sample – Potato or any other vegetables or fruits.
• Take a fresh Potato which is washed, cleaned and dried.
• Peel off the skin of the potato.
• Cut the potato into small cubes or slices.
• With the help of clean and dried Spatula, place the potato samples on the clean and dried porcelain tile.
• Add 2 to 3 drops of dilute iodine solution on the potato samples.
• Keep the slide undisturbed and observe the changes.

Observations

There will be a change in colour. A blue-black colour develops on the slice or cubes of the potato samples.

The result is positive.

According to the observation the food sample or the potato slice turned to blue-black on adding the iodine solution. This proves the presence of starch in the given plant source.

This was a simple experiment which is used to check for the presence of starch. This Iodine Test for Starch can be performed for both the liquid and solid food samples.

For more additional information about starch, carbohydrates, sources, its types or other related biological concepts and experiments, visit us @  BYJU’S Biology .

Put your understanding of this concept to test by answering a few MCQs. Click ‘Start Quiz’ to begin!

Select the correct answer and click on the “Finish” button Check your score and answers at the end of the quiz

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Hi I did not understand how the Iodene solution is made and how is it blue and black

How to Test for Starch

Last Updated: August 11, 2023 Fact Checked

This article was co-authored by Bess Ruff, MA . Bess Ruff is a Geography PhD student at Florida State University. She received her MA in Environmental Science and Management from the University of California, Santa Barbara in 2016. She has conducted survey work for marine spatial planning projects in the Caribbean and provided research support as a graduate fellow for the Sustainable Fisheries Group. There are 8 references cited in this article, which can be found at the bottom of the page. This article has been fact-checked, ensuring the accuracy of any cited facts and confirming the authority of its sources. This article has been viewed 113,676 times.

Starch tests are used to detect the presence of starch in leaves, foods, and liquids. The process is easy and can quickly tell you if a leaf has undergone photosynthesis by its starch levels or if a food or liquid contains starch. Using iodine to test for starch is a simple way to engage your students in a classroom experiment or create an educational afternoon at home.

Testing Leaves for Starch

• Although you can use any green leaf for this experiment, hibiscus leaves tend to produce better results. [2] X Research source

• A Bunsen burner is a type of gas burner that heats liquids and solids in chemical experiments.
• Your leaf will be done boiling when it has become completely soft. [5] X Research source
• Boiling your leaf will remove the waxy covering or cuticle that may prevent the entry of the iodine. [6] X Research source
• It is not necessary to turn off hot plates or electric water baths at this time. [8] X Research source

• A boiling tube is a cylinder tube of thin glass. It will be open on one end and will be able to withstand extreme temperatures. A test tube is suitable for this experiment.
• It is better to use a hot plate or water bath for this step as ethanol is extremely flammable.
• You may need to replenish your ethanol levels if the ethanol is no longer covering your leaf completely. [11] X Research source
• Your leaf is done boiling when all of the green coloring is transferred to the ethanol, leaving your leaf colorless. [12] X Research source
• Boiling your leaf in ethanol will cause it to become brittle. By adding a small amount of cold water, it will help the leaf regain a soft texture.
• You can also place your leaf in a petri dish, rather than placing it directly on the white tile. [15] X Research source
• Soaking your leaf is not necessary, but it will increase the accuracy of your results. [17] X Research source

Searching for Starch in Foods

• Lighter colored foods will work best for this experiment. [20] X Research source
• Control samples are not required but can be very useful when analyzing your results.
• You can add the iodine directly to your samples or place the iodine in the bottom of a cup, lay your sample on a paper towel, and place it on top of the iodine. [24] X Research source
• If you are unable to find an iodine solution you can also use Betadine (povidone-iodine mix), Lugol’s (a mixture of iodine and potassium), or a tincture (where the iodine is dissolved in water or alcohol). [25] X Research source

Conducting a Liquid Starch Test

• If you opted to place your liquid on a tile, add the drop of iodine directly to the liquid.

Expert Q&A

• Never allow your children to conduct this experiment unsupervised. Thanks Helpful 0 Not Helpful 1
• Always keep ethanol away from open flames, unless it is held in a test tube. [31] X Research source Thanks Helpful 0 Not Helpful 1
• Wash your hands immediately after using iodine as it can stain skin and clothing. Stains on the skin are temporary. Thanks Helpful 1 Not Helpful 0
• Always wear eye protection while handling chemicals and around boiling liquids. [32] X Research source Thanks Helpful 0 Not Helpful 1
• Always use heat-resistant gloves when dealing with open flames and hot plates. Thanks Helpful 0 Not Helpful 1

Things You'll Need

• Bunsen burner or hot plate
• 250 mL (8.5  fl oz) beaker
• Boiling tube
• Test tube rack
• A leaf to be tested
• 90% ethanol
• Iodine Solution
• Eye protection goggles
• Heat resistant gloves
• Dropping pipette
• A starchy food such as a raw potato, pasta, or bread
• A non-starchy food such as apples, cucumbers, or pure sugar.
• Small, disposable, plastic cups
• Newspaper to cover your work area
• A light-colored liquid
• Glass Rod to stir
• Eye protection

You Might Also Like

• ↑ https://practicalbiology.org/standard-techniques/testing-leaves-for-starch-the-technique
• ↑ https://www.youtube.com/watch?v=E9CSyXS3pXc
• ↑ http://www.preproom.org/practicals/pr.aspx?prID=1037
• ↑ http://brilliantbiologystudent.weebly.com/testing-a-leaf-for-the-presence-of-starch.html
• ↑ http://www.nuffieldfoundation.org/practical-biology/testing-leaves-starch-technique
• ↑ http://kitchenpantryscientist.com/starch-test/
• ↑ http://www.webexhibits.org/causesofcolor/6AC.html
• ↑ http://brilliantbiologystudent.weebly.com/iodine-test-for-starch.html

About This Article

Medical Disclaimer

The content of this article is not intended to be a substitute for professional medical advice, examination, diagnosis, or treatment. You should always contact your doctor or other qualified healthcare professional before starting, changing, or stopping any kind of health treatment.

Read More...

To test foods for starch, start by cutting your food into small slices and placing them in disposable plastic cups to keep your samples clean. Once your test foods are ready, add 1 drop of water to the cup that contains your control sample. After you control sample is ready, add 1 to 2 drops of iodine to the sample being tested, putting the iodine directly onto your sample or placing it in the bottom of the cup. Notice if your iodine has turned blue-black in color, which indicates that starch is present. If, on the other hand, your sample looks brown, there’s no starch present. For more tips from our Science co-author, including how to test for starch in liquids, keep reading! Did this summary help you? Yes No

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Microbe Notes

Leaf Starch Test: Principle, Procedure, Results, Uses

Starch in a leaf can be easily detected in a lab with the help of iodine solution. This test is called the ‘Leaf Starch Test’ or ‘Iodine Test for Starch’.

Green leaves are the food factory of plants. Green leaves have abundant chloroplasts – special organelles where the photosynthesis process takes place – so, a large portion of photosynthesis occurs in the leaves of a plant. The glucose produced during photosynthesis is stored as an energy reserve in the form of starch in the leaf, stem, branches, roots, and fruits of a plant. Starch is one of the abundant natural carbohydrates consumed in the diet by humans and other animals as an energy source.

Starch is a complex polymeric carbohydrate (polysaccharide) stored as a reserve food material in plants. It is formed of glucose monomers joined together by a glycosidic bond. The glucose units exist in two forms in natural starch; amylose and amylopectin . Amylose is water insoluble straight-chain polymer of D-glucose subunits linked together by α-1,4 glycosidic bond. Amylopectin is a water-soluble branched chain polymer of D-glucose subunits linked together by α-1,6 glycosidic bond.

Table of Contents

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Objectives of Leaf Starch Test

• To detect the presence of starch in a leaf
• To assess the extent of photosynthesis occurring in the leaf

Principle of Leaf Starch Test

Iodine is insoluble in water; but when potassium iodide is added, it dissociates into K + and I -, and the resulting I – reacts with molecular iodine (I 2 ) to form a triiodide complex (I 3 – ). The triiodide complex can further associate with molecular iodine and form pentaiodide complex (I 5 – ) and so on.

The amylose component of starch is arranged in the form of helical coils. When the iodine-iodide solution is added over starch molecules, the negatively charged polyiodide (mainly triiodide, I 3 – ) slips inside the helices of the amylose chain forming a charge transfer complex. Electrons in this charge transfer complex absorb light energy and get excited. This phenomenon is perceived by the human eye as intense blue-black color.

Hence, in the presence of starch, a blue-black colored complex is formed when the iodine-iodide solution is added over the starch. The intensity of the blue-black color is proportional to the quantity of amylose (or starch) but doesn’t give an exact quantitative (concentration) value. Hence, the test is a qualitative type test.

Requirements for Leaf Starch Test

Procedure of Leaf Starch Test

• Pluck a green leaf of any outdoor plant. A medium size leaf, preferably, a leaf recently exposed to sunlight is better for this test. 
• Boil about 250 mL water in a beaker and put the leaf in the beaker and let it boil for a few minutes (2 to 5 minutes) till its waxy coat got off and it gets soft.
• Using forceps, take out the leaf and spread it on a petri plate. 
• Place the leaf in a test tube and pour ethanol (90% or more v/v) till the leaf submerses. 
• Place the test tube in the beaker with boiling water (or in a water bath) and let the ethanol boil till the leaf decolorizes. Take out the leaf after 5 to 10 minutes if it doesn’t decolorize completely. 
• Place the leaf on a petri plate and spread it properly and rinse with cold water. 
• Using a dropper, add a few drops of iodine solution over the leaf to cover it. 
• Examine the color of the leaf after 2 minutes of the addition of iodine solution.

Observation of Leaf Starch Test

• The leaf will decolorize and become whitish after boiling in an ethanol solution. 
• The leaf will turn dark blue-black color after the addition of iodine solution. 

Result and Interpretation of Leaf Starch Test

The development of a blue-black color over the surface of the leaf indicates the presence of starch in the leaf. It suggests that the leaf was undergoing a photosynthesis process and had starch within it. 

Precautions

• Use forceps to place a leaf in and out of the boiling water and ethanol solution. 
• Always use green leaves exposed to sunlight for better results. 
• Do not direct the mouth of the test tube with ethanol towards your face while boiling it. 

Uses of Leaf Starch Test

• In the assessment of the photosynthetic activity in leaves.
• It is used to study photosynthesis patterns, starch accumulation, and depletion patterns in leaves, and assessment of environmental factors influencing photosynthesis and starch accumulation. 
• It is used as a teaching tool for basic-level students to introduce them photosynthesis process in leaves and carbohydrate storage. 

Limitations of Leaf Starch Test

• It is a qualitative test and hence only indicates the presence or absence of starch but doesn’t represent the quantity of starch present. 
• This test can be easily influenced by exposure of the leaf to sunlight, condition of the leaf, and quality and quantity of iodine solution. 
• https://www.bbc.co.uk/bitesize/guides/zpcvbk7/revision/3
• https://practicalbiology.org/standard-techniques/testing-leaves-for-starch-the-technique
• https://www.wikihow.com/Test-for-Starch
• https://chem.libretexts.org/Bookshelves/Biological_Chemistry/Supplemental_Modules_(Biological_Chemistry)/Carbohydrates/Case_Studies/Starch_and_Iodine
• https://www.biologyonline.com/dictionary/iodine-test
• https://microbiologynote.com/iodine-test/
• https://learning-center.homesciencetools.com/article/test-for-starch-photosynthesis/
• https://science.cleapss.org.uk/resource-info/pp088-testing-leaves-for-starch.aspx

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Detecting starch in food on a microscale

In association with Nuffield Foundation

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Test different foodstuffs for the presence of starch using iodine in this microscale class practical

In this experiment, students conduct qualitative tests to find out whether different foodstuffs contain starch. Working on a microscale, students produce iodine in situ by adding potassium iodide crystals and sodium hypochlorite solution to small samples of various foods. They then note any colour change to blue-black, indicating that starch is present.

A quick and easy class experiment. It should be possible to test a range of foodstuffs in about ten minutes.

• Eye protection
• Clear plastic film (eg acetate sheet as used for an overhead projector)
• Forceps (for handling foodstuffs)
• Paper towels
• Sodium hypochlorite solution, 5% w/v of available chlorine (IRRITANT), 10 cm 3
• Potassium iodide crystals, allow 5–10 small crystals per group
• A range of foodstuffs, broken into small pieces, to include both starchy and non-starch-containing foods

Health, safety and technical notes

• Read our standard health and safety guidance.
• Wear eye protection throughout.
• Sodium chlorate(I) solution (sodium hypochlorite), NaOCl(aq), (IRRITANT at concentration used) – see CLEAPSS Hazcard HC089 . Note this is NOT sodium chlorate(V), NaClO 3 . Sodium chlorate(I) solution can be purchased as such from chemical suppliers. However domestic chlorine-containing bleach solution is quite adequate for this experiment, preferably a cheap brand containing no added detergent or perfumes. Household ‘bleaches’ based on peroxide are becoming more widely available and do not contain chlorine; they should therefore not be used. The sodium chlorate(I) solution should be provided in such a way that students can add a single drop using a plastic dropping pipette. Plastic dropper bottles of capacity 30–60 cm 3 would be suitable for this purpose.
• Potassium iodide crystals, KI(s) –  see CLEAPSS Hazcard HC047b .

It is worth pre-testing the foodstuffs to check that they test correctly – that is, the starchy foods contain enough free starch to give a clear positive test, and the non-starchy foods have not been contaminated by starch-containing material. Note that the amount of free starch present in some uncooked foods may be small, and the test may work more reliably on cooked food.

Suggestions for foodstuffs for testing:

• Place a small piece of each of the foods to be tested on the plastic sheet.
• Place a small potassium iodide crystal on top of the piece of food.
• Add one drop of bleach solution (sodium hypochlorite solution) and allow it to run over both crystal and food.
• If an intense blue-black colour is seen, the food contains starch.
• Clean the plastic sheet with a moistened paper towel.

Teaching notes

The chlorine available from the bleach solution reacts with potassium iodide to form potassium chloride and iodine. The iodine then forms an intense blue-black coloured complex with any starch present. If starch is not present, only the brown colour of iodine in the presence of iodide ions will be seen. The nature of the coloured complex is beyond the level of the students, but note that it is an unstable substance from which the iodine can be easily removed by, for example, sodium thiosulfate.

Each group can be allocated a selection from the range of available foodstuffs, perhaps two starchy foods, and two non-starchy. The class results can then be pooled.

Additional information

This is a resource from the  Practical Chemistry project , developed by the Nuffield Foundation and the Royal Society of Chemistry. This collection of over 200 practical activities demonstrates a wide range of chemical concepts and processes. Each activity contains comprehensive information for teachers and technicians, including full technical notes and step-by-step procedures. Practical Chemistry activities accompany  Practical Physics  and  Practical Biology .

© Nuffield Foundation and the Royal Society of Chemistry

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Practical Biology

A collection of experiments that demonstrate biological concepts and processes.

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Practical Work for Learning

Published experiments

Testing leaves for starch: the technique, demonstration or class practical.

This procedure kills a leaf, disrupts the cell membranes and softens the cuticle and cell walls. This makes it possible to extract the chlorophyll with hot ethanol and also allows the iodine solution to penetrate the cells and react with any starch present.

Lesson organisation

You can run this as a teacher demonstration, or with students carrying out the procedure in pairs.

Apparatus and Chemicals

For each group of students:.

Eye protection

Beaker for boiling water, 250 cm 3

Boiling tube, 1 for each type of leaf used

Anti-bumping granules (optional)

For the class – set up by technician/ teacher:

Ethanol (IDA) ( Note 1 )

Kettles of boiling water ( Note 2 )

OR Electric water baths set at 90 °C containing a boiling tube rack

Iodine in potassium iodide, solution in dropper bottles ( Note 3 )

Beaker or jar (at least 250 cm 3 ), labelled ‘Waste ethanol’ ( Note 4 )

Leaves, different types, such as pelargonium (pot geranium) ( Note 5 )

Health & Safety and Technical notes

Ethanol (IDA), iodine solution and hot liquids require safety precautions to be taken. Wear eye protection.

Read our standard health & safety guidance

1 Ethanol (IDA) – refer to CLEAPSS Hazcard 40A and student safety sheet 60 – is highly flammable (flash point 13 °C) and harmful (because of the presence of methanol). The risks in this procedure are reduced by using hot water from kettles or in water baths rather than heating with a Bunsen burner flame. Some protocols recommend propanol (Hazcard 84A) in place of ethanol, as it removes chlorophyll more effectively. However, it has the additional risk of eye damage, its flashpoint is very similar to that of ethanol (IDA), and it may be more expensive.

2 Kettles are a safer source of hot water than heating with a Bunsen burner because of the presence of flammable ethanol (IDA) in this procedure. Students are familiar with the hazards of using kettles. Consider how to limit the movement of students around the laboratory with kettles or beakers of near-boiling water. Electrically-heated and thermostatically-controlled hot water baths may be a safer alternative.

3 Iodine solution – refer to CLEAPSS Hazcard 54B and Recipe card 39. A 0.01M solution is suitable for starch testing. Make this by 10-fold dilution of 0.1M solution. Once made, the solution is a low hazard but may stain skin or clothing if spilled, and may irritate the eyes.

4 Save the waste ethanol as a source of chlorophyll for future work. Make sure it cannot be tipped over and is in a safe place so it is not a fire hazard.

5 If the teacher or technician snips the leaves from the plants to give to the students, the plants are more likely to survive to be used again. Variegated Pelargonium (pot geranium) are good subjects for this experiment as are Tradescantia and Impatiens (busy lizzie).

6 Ensure that the plants have been well-illuminated for 24-48 hours. In winter, it might be worth using a halogen lamp to ensure the illumination is adequate.

Ethical issues

There are no ethical issues associated with this procedure.

SAFETY: Ensure the ethanol is kept away from naked flames. Students should wear eye protection when working with ethanol or iodine solution. Take care with hot liquids. Be aware that plant sap may irritate the skin.

Investigation a Collect leaves from the plants to be tested.

The Iodine test for Starch

In this A-Level Biology lesson "The Iodine test for starch" you will learn the procedure for testing foods for the presence of starch.

You must be able to describe the Iodine test and explain the results. For example, a colour change (Purple [blue-black] colour) indicates a positive result, staining with iodine in potassium iodide solution... 

Remember starch is a compact coiled polymer of a-glucose.

When you’re confident you can describe the Iodine test procedure and explain the results it's time for you to complete the accompanying “Iodine test for starch” lesson booklet with knowledge check and exam style questions. As in the previous lesson, you’ll be able to check your answers with mine written in the back of the work book and  see exactly how you should write your answers in a way that gains maximum marks in the exams / assignments.

A-Level Biology "The Iodine Test for Starch"

Here's a summary of the lesson: -

Starch is a complex polysaccharide composed of Amylose and Amylopectin. The combination of these two polysaccharides is the reason starchy foods are a great source of energy.

Remember, Amylopectin is branched (having both a-1,4 glycosidic bonds and a-1,6 glycosidic bonds) making it more readily hydrolysed “releasing those a-glucose molecules”. Whereas Amylose is tightly coiled and compact resulting in a much slower release of the a-glucose molecules.

The Iodine Test:

To test for the presence of Starch in a sample…

Add 10 drops of Iodine in Potassium Iodide Solution to the test sample.

Observe the results: Positive results will show a Purple [blue-black] colour.

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Science project, testing for starch.

Grade Level: 6th - 8th; Type: Chemistry

This science project will demonstrate how starches are converted to sugar during mastication and salivation.

Research Questions:

• How can you test a food for starch?
• How do chewing and salivating help with the digestion process?

Many of the foods you eat are made up of starch. Starch molecules are large, though, and need to be broken down in order to be digested. In this science project, you will examine just what happens during the salivation and chewing processes.

• Plastic gloves
• Safety goggles
• Tincture of iodine
• 2-3 identical crackers
• Several plates or saucers
• Cooked potato
• Measuring spoons
• Glucose testing strips (e.g., Diastix), optional

Experimental Procedure:

• Put on your plastic gloves and safety goggles.
• Mix 10 drops of tincture of iodine with 30 drops of water to make an iodine solution.
• Put a cracker on a plate, and test it for starch using a drop of the iodine solution.
• Chew a second cracker for 60 seconds until it is completely mixed with saliva. Spit out the chewed cracker onto another plate, and add a drop of iodine. You may be surprised to observe that the iodine no longer changed colors. The process of chewing the food and mixing it with saliva breaks down the starches in the cracker and converts them into sugars.
• Use this process to test other starchy foods, such as a cooked potato. Does chewing the cooked potato have the same effect as chewing the cracker?
• Put two small piles of cornstarch, containing ¼ teaspoon each, on another plate.
• Use the dropper to remove some saliva from your mouth. Put at least ten drops of saliva on one of the cornstarch piles and mix it around.
• Test both piles with the iodine solution. Does simply adding saliva turn the starch into sugar?
• If you’d like, you can add a teaspoon of water to each of the above foods and test them for sugar using a glucose stick (such as the type diabetics use to test their urine).

Terms/Concepts:

• Which foods contain starch?
• What are the first several steps of the digestion process?
• How are sugars and starches related?

References:

• Easy Genius Science Projects with Chemistry, by Robert Gardener. Pp 95-96, 101.

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Testing for Starch in Food

FOOD and NUTRIENTS

The earth provides us with many resources including food and fiber. Much of the food we eat comes from plants. Crops are plants that are grown specifically to be food which include grains, fruits, and vegetables.

Vitamins, minerals, carbohydrates, protein, and fat are nutrients that come from food and our bodies use to function.

Carbohydrates are the most common source of energy for the body. It is mainly provided through plant foods and includes sugars and starches. Starches are chemically bound clusters of sugar molecules found in plants. Your body breaks down starch molecules into sugar.

Testing for Starch

There is a simple test to determine if a food contains the nutrient, starch. Iodine can be used as an indicator of starch in food, because in the presence of starch, iodine makes a chemical reaction to turn the sample to a dark blue or purple black color. If there is no starch present, the iodine remains the original brownish yellow color.

Paper Towel, paper or plastic plate or protective surface

Iodine* (Be careful it can stain skin and clothes)

*Check your first aid kit or the local pharmacy for iodine.

Different kinds of food for testing such as:

Cut potatoes or potato chips or hash browns

Cut fruit like an orange or apple slice

Cheese (I used parmesan because it is light colored)

Corn or popcorn

(You can use whatever you have on hand. Just choose a variety of foods from different food groups)

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Science Projects > Life Science Projects > Test for Starch in Plants  

Test for Starch in Plants

Photosynthesis is the process in which green plants (primarily) convert energy from the sun’s light into usable, chemical energy. Plants require energy for growth, reproduction, and defense. Excess energy, created from photosynthesis, is stored in plant tissue as starch. Starch is a white and powdery substance. It houses glucose, which plants use for food. The presence of starch in a leaf is reliable evidence of photosynthesis. That’s because starch formation requires photosynthesis.

( Adult supervision required. )

Starch Testing Experiment

What you need:.

• Beaker or glass jar
• Saucepan on the stove
• Ethyl alcohol
• Iodine solution

Test for starch in plants:

1. Place one of the plants in a dark room for 24 hours; place the other one on a sunny windowsill.

2. Wait 24 hours.

3. Fill the beaker or jar with ethyl alcohol.

4. Place the beaker or jar in a saucepan full of water.

5. Heat the pan until the ethyl alcohol begins to boil.

6. Remove from the heat.

7. Dip each of the leaves in the hot water for 60 seconds, using tweezers.

8. Drop the leaves in the beaker or jar of ethyl alcohol for two minutes (or until they turn almost white).

9. Set them each in a shallow dish.

10. Cover the leaves with some iodine solution and watch.

What Happened:

The hot water kills the leaf and the alcohol breaks down the chlorophyll, taking the green color out of the leaf. When you put iodine on the leaves, one of them will turn blue-black and the other will be a reddish-brown. Iodine is an indicator that turns blue-black in the presence of starch. The leaf that was in the light turns blue-black, which demonstrates that the leaf has been performing photosynthesis and producing starch.

Try the test again with a variegated leaf (one with both green and white) that has been in the sunlight. A leaf needs chlorophyll to perform photosynthesis — based on that information, where on the variegated leaf do you think you would find starch?

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Lab Experiments to Test for the Presence of Starch When Using Potassium Iodine

How-to Science Experiments for Kids With Iodine and Cornstarch

Potassium iodide and iodine solutions are prime examples of indicators, chemicals used to identify the presence of various substances. Indicators change color when they react to a material -- in iodine and potassium iodide’s case, they react in the presence of starch. Because starch is incredibly common, these experiments with iodide solutions offer an interesting and easy way to learn about the use of indicators at home or in the classroom. Be careful using iodide solutions and do not eat food tested with it: the solutions can stain clothes and skin, and iodine can be poisonous.

TL;DR (Too Long; Didn't Read)

With a solution of potassium iodide, it’s possible to test for the presence of starches in liquids, in foods and in freshly-trimmed plant leaves -- where starches are naturally produced. Keep in mind that iodide solutions are only a qualitative indicator for starches and not a quantitative one: they can detect that starches are present, but cannot determine how much starch is present in a given substance.

Testing for Starches

Plants form starches, polymer chains of individual glucose sugar molecules, to store extra energy produced during photosynthesis. Starches come in two forms that both curve into spiral shapes: one long polymer chain known as amylose, or many individual chains attached in branching patterns called amylopectin. Solutions of potassium iodide and iodine form complex iodide ions that, while soluble in water, change color in the presence of starches -- the ions get stuck in the spirals of the starch polymer chains, forcing the iodide ions to become linear and change their electron arrangement. This causes a color change: in the presence of amylose, it becomes blue-black; With amylopectin it becomes a pale purple-red.

Testing in Solids

Before you complete any test for starch, make an iodide solution first. Dissolve 10 grams (0.35 ounces) of potassium iodide and 5 grams (0.18 ounces) of iodine in 100 milliliters (3.4 fluid ounces) of water, then stir. You can use this solution to determine what foods or natural substances contain starches -- place a few drops of the mixture on items such as chicken, potatoes, stones, cucumbers, wood, apples or pears, and watch to see if the solution changes color. If it does, the item contains starch.

Testing in Liquids

Because the complex iodide ions in the solution are soluble in water, use them to test for the presence of starches in liquids as well as in solid items. For this experiment, fill four cups with liquids: two with plain water and two with milk. Dissolve a spoonful of corn starch in one of the water cups and one of the milk cups, then add a few drops of iodide solution to each -- regardless of liquid, the solution will react to the corn starch if it’s present.

Testing for Photosynthesis

You can use an iodide solution to test leaves for starch, and determine whether the plant has performed photosynthesis recently. To do this, put one green-leafed plant in a dark closet, and another on the windowsill where it can receive sunlight. Wait a few days, then take a leaf from each of the two plants: Blanch them in hot water and submerge each leaf in ethyl alcohol until the leaves are colorless. Once the leaves are taken out of the alcohol and placed on dishes, you can use the indicator solution to determine which of the leaves came from the windowsill plant, as only it will turn blue-black.

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• Encyclopaedia Britannica: Starch
• Elmhurst College: Starch-Iodine
• Web Exhibits: Do It Yourself: Starch Test

About the Author

Blake Flournoy is a writer, reporter, and researcher based out of Baltimore, MD. Working independently and alongside professors at Goucher College, they have produced and taught a number of educational programs and workshops for high school and college students in the Baltimore area, finding new ways to connect students to biology, psychology, and statistics. They have never seen Seinfeld and are deathly scared of wasps.

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Iodine Test For Starch: Reagents, Procedure & Results Interpretation

Principle of iodine test for starch.

The starch-iodide complex as charge is transferred between the starch and iodide ions (tri-iodide or pentaiodide). The transfer of the charge between the starch and the iodide ion changes the spacing between energy levels/orbitals.  This change results in the starch-iodide complex absorbing light at different wavelength resulting in an intense color (blue black).

The intensity of the color decreases with increasing temperature and with the presence of water-miscible organic solvents such as ethanol. The test cannot be performed at very low PH due to the hydrolysis of the starch under these conditions.

Iodine test is used to test for the presence of starch in any given food sample. The test can be performed for both the liquid and solid food samples.

Reagents And Apparatus

• Test substance:  This could be a food sample, a plant extract, or any other material you suspect contains starch.
• Iodine solution:  You can buy iodine solution at a pharmacy or chemical supply store. It is usually a solution of iodine and potassium iodide (KI) in water, and it appears brownish in color.
• Pipette or dropper:  To accurately measure and transfer the iodine solution.
• Test tubes or small containers:  To hold the test substance and iodine solution.
• Bunsen burner or hot plate (optional):  If you are testing a solid substance like a potato slice, you may need heat to help release the starch for the test.

Iodine Test Procedure

• If you are testing a solid substance, such as a piece of potato or bread, you should start by creating a starch solution. To do this, cut or grind the solid material into small pieces and mix it with a small amount of distilled water. Heat the mixture slightly to help dissolve any starch. Alternatively, you can use a liquid substance, like a plant extract or a food sample, without the need for additional preparation.
• Transfer a small amount of the test substance (solid or liquid) into a test tube or a small container.
• Using a pipette or dropper, add a few drops of iodine solution to the test substance. It’s important to add the iodine solution in small increments to avoid excessive contamination of the solution.
• Observe the color change in the mixture.
• Record your observations, including the color change and any other relevant details.
• If you want to confirm the absence of starch, you can perform a control test by adding a few drops of iodine solution to a separate container with a known starch-free substance (e.g., distilled water) as a comparison.

Precautions

• Use test-tube holder for holding the test tubes and keep the mouth of the test tube away from yourself while heating.
• Use dry-clean test tubes
• Do not use too much of iodine

Result Interpretation

• Positive Result : The color of the solution changes to blue-black on addition of iodine. This indicates that starch is present in the solution.
• Negative Result : No observable color change on addition of iodine solution. This indicates that starch is absent in the solution.

Limitation of Iodine Test

• The iodine test is primarily specific to starch. It may not detect other polysaccharides or carbohydrates with different structures, such as glycogen or cellulose. Therefore, it’s essential to recognize that a positive iodine test indicates the presence of starch but not necessarily other types of carbohydrates.
• The iodine test can detect the presence of starch but may not be highly sensitive in identifying trace amounts. It’s more suited for qualitative analysis (presence or absence) than for quantitative measurements.
• Certain substances, like some dextrins and glycogen, may also produce a positive iodine test because they have similar structures to starch. In some cases, this can lead to false-positive results.
• The color change reaction with iodine solution may take some time to develop fully. It’s important to allow sufficient time for the reaction to occur before interpreting the results accurately.
• Substances in the test sample that can react with iodine or the iodine solution itself can interfere with the test results. This can lead to inaccurate readings. Therefore, it’s important to use distilled water and clean containers to minimize interference.
• The temperature at which the test is conducted can influence the results. Higher temperatures may accelerate the reaction, but extreme heat can denature the starch and affect the outcome.
• The iodine test provides limited information about the type or source of starch present. It cannot differentiate between starch from different plant sources, for example.

Practical Science

Table of Contents

Exploring Photosynthesis Variables: A Comprehensive Leaf Starch Test Experiment

Introduction:.

Delve into the fascinating world of plant biology with this comprehensive practical experiment, designed to test the effects of different variables on the rate of photosynthesis in leaves. Photosynthesis, the process by which plants convert sunlight, carbon dioxide, and water into glucose and oxygen, is vital to life on Earth. By modifying variables such as light exposure and carbon dioxide availability, we can observe how these factors impact starch production in leaves and gain a deeper understanding of the factors that influence photosynthesis. Uncover the intricacies of plant life and the essential role that photosynthesis plays in the balance of our ecosystem.

Materials and Equipment:

• Fresh green leaves from a plant exposed to sunlight for several hours (Geraniums work best)
• Aluminum foil
• Calcium oxide (quicklime)
• Test tube or boiling tube
• Forceps or tweezers
• Bunsen burner or hot plate
• Ethanol (alcohol)
• Iodine solution
• White tile or ceramic plate
• Safety goggles
• Lab coat or apron

Step-by-Step Method:

• Safety first: Put on your safety goggles and lab coat or apron to protect your eyes and clothing from potential spills.
• Choose a healthy green leaf from a plant that has been exposed to sunlight for several hours, ensuring the leaf has had ample time to undergo photosynthesis.
• Modify the variables: a. Light exposure: Cover a portion of the leaf with aluminum foil, blocking sunlight from that area and preventing photosynthesis. b. Carbon dioxide availability: Place the plant in a container filled with calcium oxide (quicklime) to absorb carbon dioxide, thereby limiting the plant’s access to this essential component of photosynthesis.
• Leave the plant under these modified conditions for a few hours.
• Boil a beaker of water on a Bunsen burner or hot plate. Use the forceps or tweezers to hold the leaf and immerse it in the boiling water for approximately 1-2 minutes. This step will soften the leaf and kill the cells, halting further photosynthesis.
• Carefully remove the leaf from the boiling water using the forceps or tweezers, and then immerse it in a test tube or boiling tube filled with ethanol (alcohol). Ensure the leaf is fully submerged.
• Place the test tube or boiling tube containing the leaf and ethanol in the beaker of hot water. The ethanol will heat up and decolorize the leaf, removing its chlorophyll. This process should take around 5 minutes. Note: Ethanol is highly flammable, so ensure there are no open flames nearby.
• Once the leaf is decolorized, carefully remove it from the ethanol using forceps or tweezers, and rinse it with cold water to remove any residual ethanol.
• Place the leaf on a white tile or ceramic plate, and add a few drops of iodine solution. The iodine will react with any starch present in the leaf, turning it a blue-black color.
• Observe the leaf for any blue-black coloration, which indicates the presence of starch. Compare the areas of the leaf that were exposed to different variables.

Safety and Troubleshooting:

• Always wear safety goggles and a lab coat or apron to protect your eyes and clothing from potential spills.
• Use caution when handling hot equipment and liquids to avoid burns.
• Ethanol is highly flammable, so ensure there are no open flames nearby when heating the ethanol.

Test Questions:

• What are the two variables being tested in this experiment, and how are they modified?
• Why is it important to cover a portion of the leaf with aluminum foil during this experiment?
• How does calcium oxide affect the rate of photosynthesis in the plant?
• What conclusions can you draw from the blue-black coloration observed in different parts of the leaf?
• Why is it important to study the effects of different variables on the rate of photosynthesis?

Answer Key:

• The two variables being tested in this experiment are light exposure and carbon dioxide availability. Light exposure is modified by covering a portion of the leaf with aluminum foil, and carbon dioxide availability is altered by placing the plant in a container filled with calcium oxide.
• Covering a portion of the leaf with aluminum foil is important because it blocks sunlight from that area, preventing photosynthesis from occurring and allowing us to observe the effects of light exposure on starch production.
• Calcium oxide absorbs carbon dioxide, limiting the plant’s access to this essential component of photosynthesis, and thus affecting the rate of photosynthesis in the plant.
• The blue-black coloration observed in different parts of the leaf indicates the presence of starch, which is a product of photosynthesis. Comparing the coloration in areas exposed to different variables helps us understand how these factors impact the rate of photosynthesis and starch production.
• Studying the effects of different variables on the rate of photosynthesis is important because it helps us understand how environmental factors can influence plant growth and productivity, which has implications for agriculture, ecosystems, and climate change.

By conducting this practical experiment, students can gain valuable insights into the factors that affect photosynthesis and explore the significance of these variables in plant biology. This hands-on approach encourages curiosity and appreciation for the natural world while reinforcing key scientific concepts.

Discovering Photosynthesis: Testing a Leaf for Starch – A Hands-On Practical Experiment

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Plain Language Summary

Introduction, statement of ethics, conflict of interest statement, funding sources, author contributions, data availability statement, relationship between international dysphagia diet standardisation initiative flow test and consistometric measures for consistency classification: an examination of thickened liquids prepared using starch-based and xanthan gum-based thickening agents.

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Mingyue Xiong , Nelson Ng , Brian Siu , Manwa L. Ng; Relationship between International Dysphagia Diet Standardisation Initiative Flow Test and Consistometric Measures for Consistency Classification: An Examination of Thickened Liquids Prepared Using Starch-Based and Xanthan Gum-Based Thickening Agents. Folia Phoniatr Logop 2024; https://doi.org/10.1159/000540118

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Introduction: Consistency of liquid food plays an important role in managing patients with dysphagia, which can be objectively evaluated by using International Dysphagia Diet Standardisation Initiative (IDDSI) Flow Test and consistometry. The present study established the relationship between IDDSI Flow Test and consistometric measures and examined the measurement limitations of each test associated with thickened liquids prepared using starch-based and xanthan gum-based thickening agents. Methods: Thirteen thickened liquid samples of consistency ranging from IDDSI level 1 (mildly thick) to level 3 (moderately thick) were prepared using starch-based and xanthan gum-based thickeners. IDDSI Flow Test and consistometric measures were obtained and analyzed using correlation and regression. Results: A strong correlation was observed between both tests. Regression analyses revealed a linear and a quadratic relationship between IDDSI Flow Test and consistometric measurements, respectively. Conclusion: Starch-based and xanthan gum-based thickeners exhibited different relationships between IDDSI Flow Test and consistometric measurements. Findings allow easy conversion and adaptation of consistometric measures to the IDDSI framework, which renders the use of consistometry in the clinical settings as a complementary quantitative measurement of liquid consistency to IDDSI Flow Test.

The study aimed to establish the relationship between the International Dysphagia Diet Standardisation Initiative (IDDSI) Flow Test (the system used by speech-language pathologists) and consistometric measures (actual measurement of consistency), as well as examine the limitations of each test when measuring thickened liquids prepared with starch-based and xanthan gum-based thickeners. These tests are important for managing patients with dysphagia, a condition that affects swallowing ability. Thirteen liquid samples with varying consistencies were prepared using both types of thickeners, ranging from mildly thick (IDDSI level 1) to moderately thick (IDDSI level 3). The researchers then analyzed the results using correlation and regression methods. Results showed a strong correlation between the IDDSI Flow Test and consistometric measures. The relationship between the two tests was found to be linear for starch-based thickeners and quadratic for xanthan gum-based thickeners. In conclusion, the study found that the relationship between the IDDSI Flow Test and consistometric measurements varies depending on the type of thickener used. This finding allows for easy conversion and adaptation of consistometric measures to the IDDSI framework, making consistometry a useful complementary tool for measuring liquid consistency in clinical settings alongside the IDDSI Flow Test.

Swallowing is a complex physiological process involving a series of highly synchronized muscle activities, facilitating the passage of liquid and solid food to the stomach [ 1 ]. Dysphagia refers to difficulties in swallowing during the transport of a food bolus from the oral cavity through the pharynx and esophagus to the stomach [ 2, 3 ]. It may stem from structural and functional deficits in respiratory, nervous, and muscular systems [ 1, 2 ]. Depending on its severity, dysphagia may lead to complications that could negatively impact an individual’s quality of life [ 4 ]. The prevalence of dysphagia has been estimated to be around 8% worldwide [ 5 ]. Recent studies reported up to 61% of the institutionalized geriatric populations in Hong Kong exhibited dysphagic symptoms [ 6 ]. Without proper and timely intervention, dysphagia may escalate to life-threatening conditions such as malnutrition, dehydration, choking, airway obstruction, penetration, and aspiration pneumonia [ 7 ].

In managing dysphagia, commercial thickening agent is often used to change the thickness or consistency of liquid food to be swallowed. It is believed the altered consistency might lead to a slower flow of liquid food and thus more time for swallowing events to happen, and in turn allowing better coordination among swallowing muscles to reduce the chance of aspiration, and eventually enhancing swallowing safety [ 7 ]. Efforts have also been made to search for natural food alternatives to commercial thickeners for cost and/or nutrition reasons [cf. 8 , 9 ]. Currently, starch-based and xanthan gum-based thickening agents are commercially available [ 10, 11 ]. Starch-based thickening agents contain modified starch granules composed of amylose and amylopectin [ 12‒14 ]. Upon contact with liquid, starch granules absorb water and swell, increasing the thickness of the mixture. However, starch-based thickeners exhibit a notable disadvantage: the resulting thickness is relatively unstable and can be influenced by external factors such as resting time, temperature, and contact with saliva [ 13‒16 ]. Although the evidence supporting the significant improvement of nutritional status and/or risk of pneumonia through the use of thickened liquids is limited [ 17‒19 ], their application remains an essential component of dysphagia management [ 20, 21 ]. Xanthan gum is a commercial biopolymer that can be used to increase liquid thickness even with a very small quantity (0.05–2%) [ 22 ]. It is widely used in bakery and salad dressing. Such thickened liquids have been shown to maintain a more stable thickness over time compared to their starch-based counterparts [ 13‒15 ]. Recent studies also reported that xanthan gum-based thickened liquids were better and safer than starch-based thickened liquids in managing dysphagic patients [ 14, 23, 24 ]. Regardless of thickener type, stability of thickened food was crucial for ensuring accurate consistency measurements. The optimal consistency for safe consumption often depends on the severity and pathophysiology of an individual’s dysphagic condition, objective measurements of liquid consistency must be established to ensure effective management.

Over the years, the use of consistometry in thickness measurement for liquid food has been common in providing standardized thickness measurements [ 25‒27 ], particularly in the food industry, in addition to other objective measurements such as fork drip test, syringe test, and ball back extrusion technique [ 24, 28 ]. A Bostwick consistometer can be used to objectively describe liquid consistency by referring to the distance the liquid traveled in the consistometer’s trough after a predetermined duration [ 29 ]. A consistometer is a simple and dependable instrument originally designed for the oil well cementing industry to measure consistency of grout [ 30 ]. Nowadays, consistometers are commonly used in the food industry to quantify consistency of viscous materials based on the distance the material flows under its own weight in a given time interval [ 31 ]. The consistometer has been used to assess the consistency of thickened drinks for individuals with dysphagia in the clinical settings [ 26, 27 ]. Researchers attempted to align consistometric measurements with traditional consistency labels such as nectar, honey, and pudding. However, practical challenges including equipment accessibility for nonprofessional stakeholders and out-patients were seen [ 5, 16 ]. The International Dysphagia Diet Standardisation Initiative (IDDSI) framework was recently developed to provide a universal standardized nomenclature for defining thickness of food and drinks, with particular emphasis on culturally sensitive terms and tool accessibility [ 32 ]. According to the IDDSI Flow Test, liquid thickness can be classified based on the amount of liquid remaining in a 10-mL BD syringe after 10 s of liquid flow [ 33 ]. Instead of relying on descriptors drawn from existing foods, consistencies are designated as levels 0 to 4, representing “thin,” “slightly thick,” “mildly thick,” “moderately thick,” and “extremely thick,” respectively.

However, the IDDSI Flow Test has limitations. For example, it may not accurately reflect IDDSI levels 3 and 4, as liquids at these thicknesses may not flow effectively in the syringe [ 34 ]. Additionally, bubbles, residue, or lumps in the liquid, which are common in drinks such as soup and freshly made juice, can lead to inaccuracies in the flow test due to blockage of the small nozzle on the syringe [ 35 ]. Classifications and reference values for different liquid consistencies associated with consistometric test and IDDSI Flow Test are shown in Table 1 .

Parameters and classifications of the Bostwick consistometer test and the IDDSI Flow Test

Although consistometer measures consistency and the IDDSI Flow Test measures thickness, these terms are often used interchangeably in the literature on dysphagia and diet modification [ 27, 36 ]. Consistometric measurements allow for assessments of more viscous liquids (IDDSI levels 3–4) and liquids with lumps, residue, or bubbles [ 25 ].

Previous research reported strengths and weaknesses associated with different measurement methods of liquid consistency [ 16, 25 ]. The IDDSI Flow Test appears to be more sensitive to thinner liquids; however, moderately and extremely thick liquids often demonstrate a ceiling effect, resulting in minimal or zero flow out of the syringe tip [ 16, 25 ]. In contrast, consistometry may not adequately measure thinner liquids for similar reasons [ 16, 25 ]. Regarding precision, the IDDSI Flow Test provides four levels with broad reference value boundaries, which could result in liquids with notably different flow behaviors being categorized as the same consistency level. The relatively finer measurable intervals in the consistometer may suggest higher measurement precision [ 27 ]. With the different classification nomenclature, the relationship between the consistometer’s measurements and IDDSI classification framework remains unclear.

Research examining the relationship between the IDDSI framework and consistometric measurements of liquid thickness is lacking. Recently, Côté et al. [ 25 ] reported a strong correlation between the two measurements; however, the data may not be interchangeable and might not fit well within a linear conversion model. Considering the differences in the underlying mechanism and complexity of non-Newtonian liquids, a nonlinear relationship might exist between IDDSI and consistometric measurements. Yet, several methodological concerns in the study by Côté et al. [ 25 ] could have influenced the validity of the findings and their applicability to institutional settings. First, the liquid samples were prepared using specific commercial products, and the composition of the thickening agent in each sample was not controlled. Variations in thickener type can significantly alter rheological characteristics of thickened liquids [ 10, 16 ], which might have potentially interacted with the testing instrument [ 25 ]. Consequently, generalizability of findings to other thickening agents is questionable. By not addressing starch-based and xanthan gum-based thickeners separately in the regression, the conclusion regarding the linearity relationship may be compromised. Furthermore, the samples were prepared using both water and juice. The reaction of thickeners to different base liquids can vary significantly due to differences in ingredient composition, pH values, and molecular weight [ 10, 37 ], making it unclear whether the results can be applied to specific types of beverages without standardization of the base liquid. Moreover, the study design by Côté et al. [ 25 ] did not align well with common clinical parameters. The consistency of thickened liquids was measured at a serving temperature of 8°C. Since the viscosity of starch-based thickened liquids is temperature-dependent [ 13, 37 ], results obtained at this single temperature point may not be transferable to room temperature. Additionally, the researchers used pre-prepared and refrigerated liquids rather than freshly mixing thickening agents with beverages at the point of consumption, which is more common in clinical settings. This introduced the variable of “setting time” into the equation. Starch-based thickened liquids are known to exhibit high instability during initial short periods and can absorb significant moisture during storage in humid environments [ 11 , 37‒39 ]. Thus, the clinical implications of the results remain uncertain. Seeing the above issues, the present study aimed to establish (1) the relationship between the thickness measurements using IDDSI Flow Test and Bostwick consistometer, (2) the measurement limitations of each test, and (3) the adaptation of consistometry to the IDDSI framework, for thickened liquids prepared using starch- and xanthan gum-based thickening agents.

The study adopted a quantitative descriptive empirical design. Ethics clearance of research was not required as no human participants were involved in the study.

Sample Preparation

To prepare the thickened liquids of different consistencies, two types of thickening agents were used: the starch-based thickening agent (ThickenUp ® , Nestlé Health Science, Switzerland) containing mainly modified food starch (maize) and the xanthan gum-based (ThickenUp ® Clear, Nestlé Health Science, Switzerland) with ingredients of xanthan gum, potassium chloride, and maltodextrin were used. They represented the most common commercial thickeners used in hospitals and elderly care centers in Hong Kong. The amount of thickening agent for preparing liquids of four consistencies conforming to IDDSI consistency labels using 200 mL of water is shown in Table 2 , as per manufacturer’s recommendations. To minimize the effect of temperature on consistency [ 13 ], all preparations were carried out under room temperature (about 25°C). To encourage reproducibility of results, thickening powder was measured in grams using a household precision electronic scale (KD-321, Tanita, Japan) (±0.1 g) instead of scoops.

Dosage of starch-based and xanthan gum-based thickening agents used to prepare 200 mL liquids of different IDDSI consistency levels

According to Cichero et al. [ 5 ], IDDSI Flow Test was most suitable for measuring consistency of liquids of slightly thick , mildly thick , and moderately thick , but not for extremely thick liquids. Therefore, only IDDSI consistency levels 1, 2, and 3 were considered when preparing thickened liquids in the present study, with an increment of 0.4 g for starch-based thickener and 0.6 g for xanthan gum-based thickener. In addition, restricted by the testing range of IDDSI Flow Test and Bostwick consistometer, with 200 mL of water, starch-based and xanthan gum-based thickeners with amount <7.2 g and <1.2 g, respectively, resulted in a ceiling effect in consistometric measures, while those with amount >12.0 g and >8.4 g, respectively, resulted in a floor effect in IDDSI Flow Test. Consequently, starch-based thickener with weights between 7.2 g and 12.0 g and xanthan gum-based thickener with weights between 1.2 g and 8.4 g, both resulting in 13 distinct levels of consistencies, were used.

To prepare for the thickened liquids, thickening powder of the correct amount was transferred to its individual beaker, then poured slowly into 200 mL of boiled tap water cooled to room temperature (∼25°C), and stirred clockwise using a fork for 1 min until no lump was present in the liquid to ensure uniformity in consistency. The thickened liquids were inspected and checked by another experimenter to make sure no lump was present. Then, the samples were set to rest for 1 min before further testing as per manufacturer’s instructions, which simulated the timeframe of ad hoc hydration needs. The entire experiment was carried out under room temperature (∼25°C).

Measurements

Iddsi flow test.

Following the recommendations by IDDSI [ 5 ], a 10-mL Luer tip syringe (Model 302143, BD™, Belgium) was used for the IDDSI Flow Test measurements. The distance from the 0 mL line to the 10 mL line was measured at 61.5 mm. To begin the measurement, 10 mL of thickened liquid was placed into an empty syringe while the nozzle was blocked with a finger to prevent leakage. Upon initiating the measurement, the nozzle was released, allowing the liquid to flow for a duration of 10 s, as timed by a digital stopwatch (A168WA-1, Casio, Japan). After precisely 10 s, the syringe nozzle was blocked once again. The remaining liquid level inside the syringe was observed at eye level, and the reading was recorded to the nearest marking (±0.2 mL) at the bottom of the meniscus to minimize error. The instrument was thoroughly washed and dried after each measurement.

Consistometric Measure

A standard Bostwick consistometer (CSC Scientific Company, Inc., Fairfax, VA, USA) was used. Made of stainless steel, the tool consists of two compartments separated by a spring-loaded trap door. One compartment is the liquid reservoir with a capacity of 75 mL, and the other compartment is a long slanted trough with 24 cm in length and 5 cm in width, with markings at every 0.5 cm interval [ 31 ]. For each measurement, the thickened sample was poured into the reservoir compartment. To ensure an exact volume of 75 mL, the reservoir was overfilled slightly, then leveled with a spatula. The spring-loaded trap door was then released to allow the fluid to flow along the slanted trough for 30 s, as timed by using a digital stopwatch (A168WA-1, Casio, Japan). After 30 s, a measurement was obtained by averaging the farthest point and the shortest point that the liquid had reached. After each measurement, the instrument was washed and dried thoroughly.

Data and Statistical Analyses

To establish the relationship between IDDSI Flow Test and consistometric measures, correlation and regression analyses were carried out, and Pearson product-moment correlation coefficients were calculated.

Average IDDSI Flow Test and consistometric measures of thickened liquids prepared using starch-based and xanthan gum-based thickening agents are shown in Tables 3 and 4 , respectively. Scatterplots were used to depict the relationship between IDDSI Flow Test measures and consistometric measures related to the use of starch-based and xanthan gum-based thickeners, and they are shown in Figures 1 and 2 , respectively.

Mean IDDSI Flow Test and consistometric measures of liquids of different consistencies prepared using starch-based thickening agent, with weight of thickener used

Mean IDDSI Flow Test and consistometric measures of liquids of different consistencies prepared using xanthan gum-based thickening agent with weight of thickener used

Relationship between mean IDDSI Flow Test and consistometric measures associated with thickened liquid prepared using starch-based thickening agent.

Relationship between mean IDDSI Flow Test and consistometric measures associated with thickened liquid prepared using xanthan gum-based thickening agent.

Correlation analyses revealed that IDDSI Flow Test measures and consistometric measures associated with starch-based and xanthan gum-based thickeners were significantly correlated ( r = −0.9812 and r = −0.9653, respectively) ( p s < 0.001). Subsequent regression analyses revealed that, for liquids thickened using starch-based thickener, a linear relationship was found between IDDSI Flow Test and consistometric measures ( R 2 = 0.9627). However, for liquids thickened with xanthan gum-based thickener, a quadratic relationship was obtained ( R 2 = 0.9834) (see Fig. 1 , 2 ). The high R-squared values associated with both thickeners indicated that, with the regression lines modeling the relationship between IDDSI Flow Test and consistometric measures, 96.27% and 98.34% of the variances were explained for starch-based and xanthan gum-based thickeners, respectively. This also indicated that the two regression lines well fitted the data obtained. The linear and quadratic regression equations obtained for starch-based and xanthan gum-based thickeners are as follows:

Relationship between IDDSI Flow Test and Consistometric Measures

The present study examined the relationship between the IDDSI Flow Test, and the consistometric measures associated with thickened liquids prepared using starch-based and xanthan gum-based thickening agents. Findings allow precise conversion of liquid consistency between both measurements and mapping consistometric measures with IDDSI Flow Test measurements. This facilitates more precise measurement of consistency of thickened liquids, which warrants safer swallowing by minimizing risks of penetration and aspiration in dysphagia management. ThickenUp ® and ThickenUp ® Clear were selected for the present study as they represented the most popular thickening agents used in Hong Kong. It was believed that both thickening agents offer stability and reliability when used to alter liquid consistency. In the study, IDDSI Flow Test and consistometric measures associated with starch-based and xanthan gum-based thickeners were measured, and regression was carried out in an attempt to obtain the equations that best fit the data points.

Starch-Based Thickening Agent

The present data revealed a linear relationship between IDDSI Flow Test and consistometric measures for thickened liquids prepared using starch-based thickener. This finding was somehow contradictory to that reported by Côté et al. [ 25 ], despite the stronger correlation found in the present study. The use of only water in the present study might explain the higher correlation found, as compared to the many types of beverages used by Côté et al. [ 25 ]. In addition, the more homogeneous samples might have contributed to greater linearity and lower variance, which in turn allowed more accurate conversion and prediction between IDDSI and consistometric measures. A linear regression equation of C = −2.33637 I + 31.146 was obtained. The negative slope in the equation implies that a higher IDDSI Flow Test value (the amount of liquid remained in the syringe after 10 s) is correlated with a lower consistometric value (the less amount of liquid flowing down the slanted trough of the Bostwick consistometer). The linear relationship between IDDSI Flow Test and consistometric measures for starch-based thickened liquid implies a proportionate increase in consistometric measure should be correlated with a reduction in IDDSI reading.

Xanthan Gum-Based Thickening Agent

Data from the experiment on xanthan gum-based thickener revealed a quadratic relationship between IDDSI Flow Test and consistometric measures. Such findings did not seem to align with those reported by Côté et al. [ 25 ]. However, it should be noted that only linear regression was used by Côté et al. [ 25 ], which could be inappropriate to reveal the relationship between two variables that are nonlinearly related and could lead to an incorrect conclusion. The nonlinear relationship between IDDSI and consistometric measures is apparent (see Fig. 2 ), and polynomial regression should be used. In the present study, the quadratic regression equation was C = − 0.183278 I 2 + 0.402632 I + 24.1862 ⁠ . According to the present finding, the quadratic relationship between IDDSI and consistometric measures implies that, for xanthan gum-based thickened liquids, the same consistometric measure may be correlated with two IDDSI readings. Future studies of a more extended range are needed to reveal a clearer picture of how IDDSI and consistometric measures are related.

Measurement Limits

Mapping between measures from IDDSI Flow Test and Bostwick consistometer was largely limited by the floor and ceiling effects of both measures. Consistometric measurements of consistency of thinner liquids prepared by either thickening agent were found to be limited by the Bostwick consistometer which had a trough of length of 24 cm. Liquids of consistency level 0 (thin) consistently exhibited a travel distance of greater than 24 cm in the 30 s of flow time, and thus was associated with a reading of >24 cm (exceeded the maximum reading of Bostwick consistometer) [ 16 ]. This can be considered as a major limitation of the present study. A longer version of the Bostwick consistometer is needed for future study that could extend and include thinner consistency. At the other end of the spectrum, the current IDDSI Flow Test failed to estimate consistency of thicker liquids. Liquids of consistency level of 4 (extremely thick) were not appropriate for IDDSI Flow Test [ 16, 25 ]. Even given 10 s to flow, the liquid simply stayed in the syringe and no liquid dripped out of it, resulting in failure in mapping of IDDSI consistency level 4. Because of such limitations, only consistency levels of 1, 2, and 3 were examined in the study (see Tables 3 , 4 ). However, using the regression equations, these consistency levels could be interpolated.

Estimation of Consistometric Measures Using Regression Equations

Based on the regression equations, consistometric measures can easily be estimated for different consistency levels. However, due to the aforementioned limitations, only IDDSI consistency levels 1, 2, and 3 could be validly estimated using these equations. According to IDDSI, consistency levels 1, 2, and 3 correspond to 1–4 mL, 4–8 mL, and >8 mL of liquid left in the syringe after dripping for 10 s (see Table 1 ). Based on this, consistometric measures corresponding to different consistency levels are estimated and shown in Table 5 .

Range of estimated consistometric measures for different consistency levels according to IDDSI framework

Limitations of the Study

The present study only examined the flow behavior of starch-based and xanthan gum-based thickened liquids using a consistometer. It is understood that both thickeners exhibit very different flow characteristics and the use of consistometer may not be able to reveal fully the flow characteristics of liquids [ 40 ]. Yet, the value of the study lies in the fact that IDDSI is commonly used by speech therapists for managing dysphagic patients, and consistometry is simple to use and to understand.

Apart from the limitations stated above, several other limitations in the present study can be identified. First, thickened liquids were prepared using only water as the dispersion medium, and only ThickenUp ® and ThickenUp ® Clear, both of which were manufactured by Nestlé Health Science, were used. This might have limited the generalizability of thickened liquids consumed by dysphagic patients. Another limitation of the study relates to the use of only linear and quadratic regressions to describe the relationship between IDDSI and consistometric readings for starch-based and xanthan gum-based thickeners, and the regressions were based on limited amount of data. Other higher order regression may be tried in relating such relationships using a large scale of data.

A third limitation of the study is the apparent floor and ceiling effects of both IDDSI and consistometric measurements, which can be found in Figures 1 and 2 . Data should therefore be interpreted with caution. In addition, other aspects of liquid food including acidity, serving temperature, and texture were not considered. Future studies involving more base liquids and other thickening agents should be carried out in order to obtain a wider picture of the association between IDDSI Flow Test measures and consistometric measures.

The present study revealed a very strong correlation between liquid consistency measures obtained using IDDSI Flow Test and Bostwick consistometer. In addition, according to regression analyses, starch-based and xanthan gum-based thickeners were found to exhibit a linear and quadratic relationship between IDDSI Flow Test and consistometric measures, and the regression equations were C = − 2.33637 I + 1.146 and C = − 0.183278 I 2 + 0.402632 I + 24.1862 ⁠ , respectively. Seeing the limited range of consistency seen in the study, future studies are needed to better map consistometric measure to IDDSI levels, especially for liquids of IDDSI levels 3 and 4.

No ethics were required for the study as it did not involve human or animal participants.

The authors have no conflicts of interest to declare.

The project was funded by the Education Faculty Research Fund, Faculty of Education, University of Hong Kong (Reference No. 000250337).

Mingyue Xiong: substantial contributions to the conception and design of the study. Nelson Ng and Brian Siu: data collection, preliminary analysis, and preliminary preparation of the manuscript. Manwa L. Ng: substantial contributions to the conception and design of the study, administration of the project, data and statistical analyses, and manuscript preparation.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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• Published: 13 August 2024

Effects of biodegradation of starch-nanocellulose films incorporated with black tea extract on soil quality

• Elham Malekzadeh   ORCID: orcid.org/0000-0002-1662-5369 1 ,
• Aliasghar Tatari 2 &
• Mohammadreza Dehghani Firouzabadi 2  

Scientific Reports volume  14 , Article number:  18817 ( 2024 ) Cite this article

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Metrics details

• Biochemistry
• Biogeochemistry
• Engineering
• Environmental sciences

This study aimed to investigate the biodegradation behaviour of starch/nanocellulose/black tea extract (SNBTE) films in a 30-day soil burial test. The SNBTE films were prepared by mixing commercial starch, nanocellulose (2, 4, and 6%), and an aqueous solution of black tea extract by a simple mixing and casting process. The chemical and morphological properties of the SNBTE films before and after biodegradation were characterized using the following analytical techniques such as field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDX), and fourier transform infrared (FTIR). The changes in soil composition, namely pH, electrical conductivity (EC), moisture content, water holding capacity (WHC), soil respiration, total nitrogen, weight mean diameter (MDW), and geometric mean diameter (GMD), as a result of the biodegradation process, were also estimated. The results showed that the films exhibited considerable biodegradability (35–67%) within 30 days while increasing soil nutrients. The addition of black tea extract reduced the biodegradation rate due to its polyphenol content, which likely resulted in a reduction in microbial activity. The addition of nanocellulose (2–6% weight of starch) increased the tensile strength, but decreased the elongation at break of the films. These results suggest that starch nanocellulose and SNBTE films are not only biodegradable under soil conditions but also positively contribute to soil health, highlighting their potential as an environmentally friendly alternative to traditional plastic films in the packaging industry.

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Introduction.

In biodegradable packaging, biopolymers such as starch, chitosan, and cellulose are obtained from renewable raw materials such as agricultural waste or food processing waste 1 , 2 , 3 . Starch is a complex polysaccharide found in all plant tissues. It consists of linear chains of α- d -glucose units linked by α(1–4)-glycosidic bonds. These chains also occasionally contain α(1–6) glycosidic links, which give the starch its branched structure 4 , 5 . The hierarchical organization of starch gives it a distinct semi-crystalline structure that influences its physical and chemical properties as well as its ability to be digested by enzymes 6 . The macromolecular nature of starch makes it an important resource in several industrial sectors such as food, pharmaceuticals, and packaging 7 , 8 .

Starch is a suitable candidate for the production of biodegradable films for sustainable packaging due to its renewable source, biocompatibility, and inherent biodegradability 1 , 2 , 9 , 10 , 11 . In packaging applications, starch can be fabricated into films with desirable mechanical properties and barrier properties by utilizing processing methodologies such as casting 9 or extrusion 12 . Further, the need for biodegradable alternatives to plastic pollution emphasizes the need for starch-based films, which are promising contenders because of their biodegradability 13 , 14 . Starch-based films are biodegradable, but they are also flexible, allowing them to be tailored to specific packaging needs by incorporating additives or blending with other biopolymers 9 , 15 , 16 , 17 . Therefore, the investigation and improvement of packaging films made from starch show great potential in tackling the increasing environmental issues linked to traditional plastic packaging 18 , 19 .

Cellulose is the most abundant organic compound in the world 20 . Nanocellulose is characterized by the deconstruction of the hierarchical arrangement of cellulose chains at the nanoscale, resulting in a unique chemical makeup 21 , 22 , 23 . The cellulose chains are produced into nanocellulose by mechanical, biological, or chemical processes 24 , 25 , 26 , 27 . Nanocellulose exhibits both crystalline and amorphous areas 28 . Nanocellulose particles possess remarkable mechanical capabilities, characterized by their outstanding tensile strength, Young’s modulus, and flexibility 24 , 28 , 29 . These qualities are attributed to their high aspect ratios and surface areas-to-volume ratios. In addition, nanocellulose has a high level of reactivity as a result of the many hydroxyl (−OH) groups present in the cellulose chains 30 . This reactivity enables easy chemical modification and functionalization, allowing for the customization of its features to suit a wide range of applications, including the production of biodegradable packaging films.

Black tea extract, obtained from the leaves of the Camellia sinensis plant, is a highly abundant source of polyphenols, specifically catechins, and theaflavins, which exhibit strong antioxidant properties 31 , 32 . These compounds not only contribute to the characteristic flavour and colour of black tea, but also offer potential benefits for packaging films 11 , 17 , 33 , 34 , 35 , 36 . Incorporating tea extract into packaging films can impart antioxidant activity, extending the shelf life of packaged products by delaying oxidation and microbial degradation 11 , 15 , 16 , 17 , 37 , 38 , 39 , 40 , 41 , 42 . Furthermore, the polyphenols in black tea extract have been shown to exhibit antimicrobial properties 43 , inhibiting the growth of spoilage microorganisms and pathogens, thereby enhancing the safety and quality of packaged goods 44 , 45 . Moreover, the natural origin of black tea extract aligns with the growing consumer demand for sustainable and eco-friendly packaging solutions 46 . By using the antioxidant and antimicrobial properties of tea extract, packaging films can offer enhanced protection and preservation of food products while reducing the need for synthetic additives or preservatives 10 , 16 , 34 , 35 , 37 , 38 , 47 , 48 . Therefore, the incorporation of tea extract in packaging films represents a promising path to develop functional and sustainable packaging materials with value-added for the food industry 49 , 50 .

The reinforcement of biopolymer packaging films with new materials is very important. Considering the increasing importance of renewable energy and the need to replace many plastics in the packaging industry, reinforcing biopolymer films with new materials can significantly improve the physical properties and performance of these films. For example, the use of plant extracts, nanocellulose and starch derived from various plants as additives in biopolymer films can act as a natural reinforcement and improve the mechanical, tensile, and moisture-resistant properties of these films, as well as increase their stability and useful life. In addition to increasing the physical properties, these additives can also significantly improve erosion resistance, gas permeability and heat resistance, which is very important for packaging applications. On the other hand, these new materials can help to fulfill the environmental goals of the packaging industry, because they use biological and renewable resources and reduce the amount of plastic waste production. In recent years, research related to black tea extract, nanocellulose, and starch in the field of production and reinforcement of biopolymer packaging films has grown significantly. Andrade et al. 49 investigated polylactic acid (PLA) films loaded with polyphenol extracts from green tea and rosemary. They reported that these active PLA packaging can contribute to the delay of lipid oxidation in foods with high-fat content. Carrizo et al. 50 reported that active food packaging based on green tea extract can have antioxidant capacity compared to control (without green tea extract). Homthawornchoo et al. 38 investigated the effects of incorporating green tea extract (GTE) into rice starch-pectin (RS-P) blend films. The addition of GTE improved the films' antioxidant properties and increased their thickness. However, it also decreased transparency, moisture content, and water vapor transmission rate (WVTR). GTE weakened the films but showed some antimicrobial activity against Staphylococcus aureus. Medina‐Jaramillo et al. 40 investigated the use of green tea and basil extracts as natural plasticizers in cassava starch-based biofilms for food coatings. They reported that green tea and basil components interact strongly with starch molecules to increase their molecular mobility, which leads to plasticization. As a result, the films were thermally stable and retained high transparency at 250 °C. Yuan et al. 48 investigated the development of active food packaging films using biopolymers derived from shrimp shell waste protein (SSWP) and chitosan (C). The film characteristics were enhanced by incorporating oolong tea extract (OTE), maize silk extract (CSE), and black soybean seed coat extract (BSSCE) at different concentrations (1, 3, and 5%, w/w). They found that adding OTE, CSE, and BSSCE significantly impacted the films physicochemical properties. Film thermal stability improves with increasing extract concentration. Rodrigues et al. 39 reported the effect of incorporation of GTE, ginger essential oil (GEO), and nanofibrillated cellulose (NFC) on the properties of starch films. They concluded that reinforcing the films with nanofibrillated cellulose improved their strength, and reduced water solubility and WVTR. The natural extracts, on the other hand, increased antioxidant activity and inhibited the growth of bacteria. Rajapaksha and Shimizu 10 found that by incorporating microencapsulated spent black tea extract (SBT), active films were improved mechanically and antioxidant. Compared to the control starch film, both types of active films were better at blocking UV light and water vapour and retarding lipid oxidation for 35 days. In a study by Perazzo et al. 16 , active films made from cassava starch bio-based and infused with aqueous green tea extract and palm oil colourants maintained butter quality for 45 days. They recommended avoiding high concentrations of green tea extract as additives to films due to its high polyphenol content, which can act as a pro-oxidant agent. A study conducted by Peng et al. 47 found that GTE significantly reduced water vapor permeability and increased film antioxidant ability. GTE films were able to scavenge DPPH radicals more effectively than black tea extracts (BTE) films across all food simulators (0, 20, 75, and 95% EtOH).

The literature review showed that research on starch films containing tea extracts is promising, but some areas could benefit from further investigation. While studies are investigating different concentrations of tea extracts, there are insufficient and comprehensive studies on starch films containing black tea extract and nanocellulose. Further research is needed on how these films behave during prolonged storage. Some studies have looked at green or black tea extracts. A closer look at the specific bioactive components responsible for the effect could lead to more targeted film formulations. Most research has focused on the properties of starch films with tea extracts for food packaging, while the behaviour of biodegradation on soil respiration and nutrients has not been studied.

In investigating the biodegradation process of SNBTE films in soil, there may be a research gap regarding the extended effects of these films on soil microbial communities and nutrient cycling. Current research is mainly focused on the degradation rate and structural durability of these films. However, there is limited knowledge of their effects on soil microbial activity, such as respiration rates and microbial biomass, and on nutrient dynamics, such as changes in carbon, nitrogen and phosphorus availability in the soil ecosystem. Addressing this research gap would provide important insights into the ecological consequences of using these biopolymer-based films as sustainable packaging materials. It would also contribute to the development of more thorough life cycle assessments to evaluate their environmental impact. This study aims to evaluate the medium-term degradation of SNBTE films in soil and to investigate the changes in film integrity, microbial activity, and nutrient availability over time. By evaluating soil respiration rates as an indicator of microbial activity and monitoring changes in soil nutrient content, including carbon (C), nitrogen (N), and phosphorus (P), this study will investigate the interactions between biodegradable films and soil ecosystems (Fig.  1 ). These findings can be used to improve packaging design and use in sustainable applications by providing a better understanding of these materials' environmental behaviour.

Schematic of the conceptual design of the research.

Material and methods

Corn starch (72 wt.% amylopectin and 28 wt.% amylose) and glycerol as a plasticizer (food grade, purity 99%) were purchased by Merck (Darmstadt, Germany) and used without further purification. Unblended and dried black tea was obtained from a local market in Ashkevarat city, Gilan province. Distilled water was used in all experiments.

Preparation of black tea extract (BTE)

The aqueous BTE was prepared following the previous report, with slight modifications 10 , 33 , 38 , 39 , 40 , 51 . In brief, aqueous extract was obtained by dispersing 5.0 g of dried black tea in 100 mL of distilled water at 100 °C for 40 min. Immediately, the hot extract was cooled to room temperature and filtered twice (Whatman No.1 filter paper). The solution was kept in dark containers at ~ 5 °C until further use.

Preparation of nanocellulose

The nanocellulose gel (2.5 wt%) was prepared from commercially bleached softwood pulp. To produce nanocellulose, water slurry containing cellulose fibres at 1.0 wt% was passed three times through a disk grinder (MKCA6-3; Masuko Sangyo Co., Ltd., Japan) 52 , 53 , 54 . To concentrate the gel, a Sigma KS centrifuge apparatus (Sigma Co., Germany) was used at 14,000 rpm for 30 min 26 .

Preparation of films (casting method)

Corn starch powder (5.0 g) and glycerol as plasticizer (1.5 g) were dissolved in 100 mL distilled water with constant stirring (65 ± 2 °C for 15 min) to achieve complete gelatinization 9 . To prepare SNBTE films, three different concentrations of nanocellulose (2, 4, and 6 wt% of starch) were added to a partially gelatinized starch solution with stirring to ensure uniform dispersion of nanocellulose in the film matrix. In addition, 2 wt% BTE was incorporated into the film solution while maintaining stirring at 65 °C for 15 min. For film formation, 25 g of each gel was poured onto 10 cm plastic Petri dishes and then immediately dried in a drying oven at 60 °C for 8 h. After drying, the films were conditioned at 23 °C and 50% relative humidity (RH) for at least 48 h before characterization.

Characterization

Mechanical properties.

The tensile properties were determined based on the American Society for Testing and Materials (ASTM) D882-18 standard, using a SANTAM universal tensile machine (model STM-1, Santam Co., Tehran, Iran) with a load cell of 1 kN and cross-head speed 10 mm/min. Film samples were cut in dumbbell shapes and mounted between the tensile grips. Three samples from each film were conditioned at temperature = 23 °C and RH = 50% for at least 48 h before testing. Tensile parameters including tensile strength and elongation at break were reported.

Morphology (FESEM/EDX analysis)

The surface morphology of the produced films was examined using field emission scanning electron microscopy (FESEM, MIRA3 TESCAN-XMU, Kohoutovice, Czech Republic) and energy-dispersive X-ray analysis (EDX, SAMx Numerix, Levens, France). The samples were dried at 40 °C for 12 h and mounted on aluminum stubs using double-sided carbon tape. Then, samples were coated with gold using a sputter coating machine (Quorum, Q150R ES, UK). The micrographs were captured at an accelerating voltage of 10.0 kV.

X-ray diffraction (XRD)

XRD is an extensively employed technique for characterizing various substances and determining their crystallinity. The X-ray characterization was performed using a diffractometer XRD (Unisantis XMD300 model, Singapore) equipped with monochromatic Cu-Kα radiation at 50 kV, 30 mA, and a sampling width of 0.02 degree. Scan values varied between 10 and 60 (2 theta).

Fourier transform infrared (FTIR) spectroscopy

The FTIR is a technique used to analyze the chemical composition of materials and identify and quantify different compounds by measuring the absorption of infrared light. FTIR spectrometers (Perkin-Elmer, Spectrum RX I) were used to examine the functional groups and chemical changes of films. A total of 64 scans were acquired with a resolution of 4 cm −1 , covering the range from 4000 to 500 cm −1 .

Water hold capacity (WHC) and moisture content

The WHC of soil was determined according to Tai et al. 55 study. Briefly, the soil was mixed with 20.0 g of distilled water, and the excess water was drained. The soil was subsequently saturated and deposited on filter paper that had been pre-dried to a constant weight. The saturated weight (W sat ) was then recorded. A dry weight (W dry ) was determined by drying the sample at 105 °C until it reached a constant mass, and the WHC was calculated by using Eq. ( 1 ). The initial weight (W ini ) of each soil sample was recorded on a previously dried filter paper. Subsequently, the samples were desiccated in an oven set at 105 °C until their weight remained constant, yielding the final soil mass (W fin ). The soil's moisture content was determined utilizing Eq. ( 2 ).

Soil respiration

In soil respiration, carbon dioxide (CO 2 ) is produced as a result of metabolic processes. As a consequence, soil respiration represents biological activity. With the excess sodium hydroxide (NaOH) titrating technique, soil respiration was measured every 10 days for 30 days 9 .

Biodegradability test

The biodegradability of films was evaluated using a slightly modified version of previously established techniques for soil burial 9 , 56 , 57 . The physical and chemical properties of the soil were determined as follows: EC (0.34 ds/m), pH (7.10), total organic carbon (0.35%), total nitrogen (0.014%), saturation point (44.0%), field capacity (35.32%), MDW (0.02 mm), GMD (1.03 mm), and texture (silty-clay-loam). To test the biodegradability of the films, 100 g of soil was mixed into a 250 mL beaker. Samples were cut (40 mm 2 ) and buried in the soil mixture in the beaker. The soil moisture was consistently kept at 27 °C and 70% of field capacity (70% FC). Dry samples were buried for 10–30 days in the wet soil of the test medium. Visual changes were occasionally observed in these samples. Following the removal of the film samples from wet soil after 10–30 days, they were repeatedly rinsed with water to remove soil adhering to the surfaces. In biological degradation testing, films were dried at 60 °C for 2 h until their weight became constant. The weight loss was calculated by using Eq. ( 3 ).

Carbon analysis

In this study, the contents of total organic carbon (TOC), cold water extractable organic carbon (CWEOC), and hot water extractable organic carbon (HWEOC) were determined. TOC was measured using both the original and modified Walkley–Black (WB) dichromate method 58 . The CWEOC and HWEOC determining was performed according to the method described in the Hamkalo and Bedernichek 59 report.

Total nitrogen

The total nitrogen (N) content of the soil was determined using the Kjeldahl method, a widely recognized and reliable technique for measuring nitrogen levels in various samples. This method involves digesting the soil sample with concentrated sulfuric acid and a catalyst, which converts organic N into ammonium ions. The ammonium ions are transformed into ammonia gas by heating and distillation. The ammonia is then titrated with a standardized acid solution to determine the N content of the sample 60 .

pH and electrical conductivity (EC)

Soil pH was assessed with a digital pH meter (model PH700 Benchtop pH Meter). The electrical conductivity (EC) of the soil was evaluated using an EC meter (Hanna Instruments, Model HI5321-02) on an aqueous soil extract.

Mean weight diameter (MDW) and geometric mean diameter (GMD)

Aggregate stability was measured according to MWD using Eq. ( 4 ) 61 , and geometric mean diameter (GMD) was measured using Eq. ( 5 ) 62 .

where, \(\overline {Xi}\) is the mean diameter of aggregates for any particular size range; W i is the weight of the aggregates in a size range as a fraction of the total dry weight of the sample; and n is the number of sieves.

where, W i is the weight of the aggregates in a size range of average diameter Xi; and ∑ is the total weight of the soil sample.

Statistical analysis

The data obtained from the investigations underwent analysis of variance (ANOVA) utilizing the SPSS 24.0 software (IBM, Armonk, NY, United States; https://www.ibm.com ).

Results and discussion

Nanocellulose characterization.

The physicochemical and morphological properties of the nanocellulose used in this study, including FESEM micrographs, diameter distribution histogram, EDX analysis, XRD diffraction pattern, and chemical analysis, are presented in Fig.  2 . The diameter of the nanofibers was determined by analyzing FESEM micrographs (N = 170) with Digimizer image analysis software (version 4.1.1.0; MedCalc Software, Mariakerke, Belgium; http://www.digimizer.com ), revealing a fibrillar structure with an average diameter of 31 ± 7.60 nm (Fig.  2 a,b). The diameter of the nanocellulose used in this study was similar to other nanofibers obtained from different sources, such as softwood fibre 9 , 63 , canola straw 28 , sugarcane bagasse fibre 52 , and waste paper industry 64 . To further understand the nanocellulose properties, the localized elemental information of nanocellulose was determined by EDX as exhibited in Fig.  2 c, which contains intense signals of C, N, and O. The XRD pattern of the as-prepared nanocellulose is presented in Fig.  2 d. The absence of significant diffraction peaks in the whole pattern indicates that the nanocellulose generated is predominantly crystalline. The peaks were observed at 2ϴ of 15.1, 16.2, 22.4, and 34.1 degrees, indicating the presence of cellulose type Iβ 65 . The chemical composition values of nanocellulose used in this study are provided in Fig.  2 e. The high amount of holocellulose and the low amount of lignin indicate the confirmation of the quality of the raw materials for the production of nanocellulose. Also, the amount of less ash content indicates the minimum mineral impurities in the produced nanocellulose.

Morphological and chemical characteristics of nanocellulose used in this research: ( a ) FESEM micrographs, ( b ) diameter distribution histogram, ( c ) EDX spectrum, ( d ) XRD diffraction pattern, and ( e ) chemical analysis.

The results for the mechanical properties of the starch/nanocellulose/black tea films are shown in Table 1 . The addition of the tea extract decreased the values for tensile strength and elongation at break compared to the films without extracts. The results showed a significant increase in the tensile strength of the films with the addition of nanocellulose. The maximum tensile strength was achieved when 6.0 wt% nanocellulose was incorporated into the SNBTE films. The tensile strength was increased to 10.54 MPa, an improvement of 40.4% compared to the control. This result was supported by Ali et al. 66 who found that the addition of nanocellulose from sugarcane bagasse increased the tensile strength but decreased the elongation at the break of PVA/starch composite films with nanocellulose. Othman et al. 67 reported that the addition of 1.5% nanocellulose (in weight percent of starch) increased the tensile strength but decreased the elongation at the break of corn starch films. Hafid et al. 68 found that the use of surface-modified cellulose from glossy paper waste in starch-based films increased tensile strength.

The cellulose-based nanomaterials possess a large specific surface area and exhibit good dispersion. These characteristics enhance the interfacial bonding between the filler and matrix, facilitating the efficient transfer of stresses from the matrix to the particles. As a result, the mechanical properties of the film are improved 69 . Because the cellulose fibers in the films were nanosized and could effectively make contact with the corn starch matrix, they promoted high intermolecular forces, increasing the tensile strength of the films containing nanocellulose. These forces led to an increase in the stiffness of the films and thus to a high tensile strength. In addition, the entanglement of the nanocellulose within the starch polymer chains may have contributed to the increase in tensile strength 66 , 67 . The strong interactions between the matrix and filler prevent the SNBTE films from elongation.

The decrease in tensile strength and elongation at break in starch/nanocellulose films incorporated with black tea extract is primarily attributed to the disruptive effects of the tea extract on the intermolecular interactions within the film matrix. The incorporation of tea extract alters the structural integrity of the film, possibly through interactions with bioactive compounds such as polyphenols and antioxidants contained in the extract. These interactions can lead to changes in the morphology and composition of the film, ultimately affecting its mechanical properties. In addition, the presence of tea extract can lead to new interactions between the film components that further influence the overall mechanical behaviour of the film. Rodrigues et al. 39 found that reinforcement with nanocellulose (2%) resulted in a significant increase in tensile strength (47.6%) and lower elongation compared to the control (starch films). Peng, et al. 47 reported that the use of green tea and black tea extract in chitosan packaging films decreased tensile strength and elongation at break. Similar results have been found in PLA and starch films incorporated with rosemary extracts 49 , 70 and thymol 67 . Martins et al. 15 assessed the tensile strength in the longitudinal direction of an active PLA film enhanced with green tea extract. They produced a control PLA film exhibiting a higher tensile strength of 40.2 MPa, which was reduced to 35.4 MPa upon incorporating 2% green tea extract.

Biodegradability

In the evaluation of film degradation in soil, the biodegradable properties of the films were examined when they were buried. The percentage of biodegradation was determined using Eq. ( 3 ) and the resulting data is shown in Table 2 . After 30 days in the soil, the control film lost 66.66% of its weight. The addition of nanocellulose and tea extract significantly (p < 0.05) reduced the amount of biodegradability of the films in the soil compared to the control. The addition of 6% nanocellulose to the starch films reduced the biodegradability by 41.67% compared to the control treatment. When films were treated with tea extract (starch-6% nanocellulose-tea extract), this value was 47.22% compared to the control treatment. The appearance changes of the films after 30 days of burial in soil are presented in Fig.  3 .

Photographs of film subjected to biodegradation in soil for 30 days. ( a ) control (pure starch), ( b ) starch-2% nanocellulose, ( c ) starch-4% nanocellulose, ( d ) starch-6% nanocellulose, ( e ), starch-2% nanocellulose-tea extract, ( f ), starch-4% nanocellulose-tea extract, and ( g ) starch-6% nanocellulose-tea extract.

The change in the appearance of the film was due to the amount of starch and glycerol in the formulation 57 . The decrease in the percentage of biodegradability of starch-nanocellulose films compared to control films (starch-only films) can be attributed to several interrelated factors. Firstly, the incorporation of nanocellulose into the film matrix alters its physical and mechanical properties, potentially resulting in reduced accessibility of microorganisms and enzymes responsible for biodegradation. Nanocellulose, owing to its high aspect ratio and surface area, can form a reinforcing network within the film, leading to increased crystallinity and reduced porosity, which may impede the diffusion of water and microbial degradation agents. Additionally, the compatibility between starch and nanocellulose phases, as well as the dispersion of nanocellulose within the matrix, can influence the overall degradation process. Chemical interactions between starch and nanocellulose may result in the formation of intermolecular bonds that render the composite film less susceptible to enzymatic attack and microbial degradation compared to pure starch films. Malekzadeh, et al. 9 reported that the starch films reinforced with nanocellulose and nano lignocellulose compared to the control sample (pure starch) had lower weight loss under soil burial conditions. Trujillo-Hernández et al. 57 reported that starch films underwent degradation, with an 83.03% weight loss observed after 15 days of exposure to soil. The samples containing 0.05% and 1% coconut bagasse cellulose had a weight loss of 82.85% and 83.65%, respectively at the same time and the samples needed more time for complete degradation in the soil compared to control. Zhang et al. 71 stated that regenerated cellulose films underwent complete decomposition into CO 2 and water by soil microorganisms within 2 months, with reported half-lives ranging from 30 to 42 days. Following 16 days of decay, the soil microorganisms reduced the film's molecular weight by 38–40%, accompanied by a 10–15% weight loss.

The decrease in percentage biodegradability observed in starch-nanocellulose films incorporated with tea extract compared to the control films and the starch nanocellulose films can be attributed to several factors. First, the addition of tea extract may alter the structural integrity and composition of the film matrix, potentially hindering the accessibility of microorganisms and enzymes responsible for biodegradation. Tea extract contains polyphenolic compounds that are known for their antioxidant properties and may act as inhibitors of microbial activity by scavenging free radicals and interfering with the enzymatic reactions involved in the degradation processes. In addition, the presence of tea extract can alter the balance between hydrophilicity and hydrophobicity of the film surface, affecting microbial adhesion and colonization and thus hindering the kinetics of biodegradation. Furthermore, the chemical interactions between tea extract components and film constituents could lead to the formation of more stable complexes, rendering the films less susceptible to enzymatic attack and microbial degradation. Similar results have been obtained by previous researchers 9 , 55 , 57 .

Morphological studies

The FESEM micrographs of film samples before and after biodegradability in soil are presented in Figs.  4 and 5 , respectively. The control films exhibited a notably smooth and flat surface morphology, with no detectable presence of starch particles (Fig.  4 ). The FESEM micrographs after biodegradation under soil burial conditions showed degradation on the surface of the film. The observed morphological shift occurred on the 30th day under soil burial conditions. The control film displayed the most extensive biodegradation, undergoing complete fragmentation. This led to the formation of significant holes and cracks on the polymer surface, evident throughout the biodegradation process. These changes are attributed to microbial activity or biofilm formation on the polymer surface, along with the diffusion of cells into the polymer's core, resulting in surface erosion and the formation of cavities within the films. The micrograph illustrates the presence of fungal spores and mycelia invading the surface (Fig.  5 ).

FE-SEM images of the film surface before biodegradation.

FE-SEM images of the film surface after biodegradation under soil burial conditions.

The fractures and pores observed on the surface in the films are similar to those reported by Trujillo-Hernández et al. 57 , in a film composed of starch, glycerol, and cellulose, which were degraded in 15 days. Also, similar results have been reported by Kalita et al. 72 regarding the formation of holes and cracks in Poly(lactic acid) (PLA)-based green biocomposites surface. Batista et al. 73 reported many changes (corrosion, cracks, and discoloration) after 2 months of biodegradation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/peach palm particles biocomposites. The weak adhesion between the fiber/PHBV interface, leading to increased water absorption and enhanced accessibility for soil microorganisms, was proposed as a contributing factor to the deterioration. In a study, Zhang et al. 71 observed a porous structure with fungal mycelium on the decayed surface in the FESEM images of regenerated cellulose films. They concluded that the biological degradation of the films was initiated by microorganisms and proceeded gradually over time.

The degradation of the material is caused by the proliferation of microbes on both the surface and interior 57 . In the microbial degradation process, extracellular enzymes are secreted to depolymerize the biopolymers 74 . During the biodegradation process, microbes first assimilate short chains, after which they proceed to attack long crystalline chains through a chain scission mechanism 72 . Direct contact with the soil facilitates the ability of microorganisms to locate carbon sources and initiate their metabolism, resulting in the reduction of weight in biofilms 9 , 57 . Starch-based materials are broken down by the process of bond cleavage, which occurs as a result of biological oxidation or biological hydrolysis and produced by microbial activity. Soil burying erosion is a physical process that involves the swelling, diffusion, and dissolving of monomers 55 . Biodegradation is the process of physical decomposition where various bacteria bind to the surface of organic materials by releasing adhesive compounds. Polysaccharides and proteins are adhesive molecules that can infiltrate materials and modify their volume, size, and pore distribution, as well as their moisture content and thermal conductivity 9 . Under regulated conditions of aerobic and anaerobic environments, microbial breakdown modifies the strength and color of polymeric material. Throughout this procedure, bacteria transform the insoluble biopolymer into a soluble biopolymer. Microorganisms utilize these chemicals in their metabolic processes for microbial growth. This conditions elucidates the process of biopolymer degradation in the presence of microorganisms, highlighting the resulting by-products such as carbon dioxide (CO 2 ), water, inorganic compounds, biomass, and methane (CH 4 ) 74 . In a similar study, Tai et al. 55 explained a similar mechanism for starch-polyurethane hybrid films. Figure  6 , presents a summary of the biodegradation mechanisms in starch-based films under soil environment.

Schematic diagram illustrating mechanisms and degradation behavior of the starch-based films under soil environment .

Enzymatic degradation drives biodegradation by catalyzing the depolymerization of polymeric chains within a matrix. This process involves hydrolase enzymes facilitating the cleavage of chemical bonds prevalent in biodegradable bioplastics, by adding water molecules. Key enzymes like cellulases and amylases target cellulose and starch polymers, respectively, while esterases and lipases act on co-polyesters, breaking ester bonds. Factors such as surface morphology, crystallinity of biomaterials, composition mixing, burial depth (influenced by microbial and environmental variations), and duration of exposure to soil collectively influence the degree of biodegradability 75 .

FTIR analysis

The FTIR spectra of various films before and after biodegradation under soil burial conditions (30 days) are provided in Fig.  7 . In the FTIR spectrum of pure starch, nanocellulose, starch-nanocellulose, and SNBTE films, different peaks can be recognized that indicate the chemical and structural properties of these materials. In pure starch, peaks associated with hydroxyl functional groups (–OH) and carbonyl groups (C=O) can be recognized. In pure nanocellulose, peaks consistent with the structural features of cellulose are observed, including C–O–C and C–H peaks. When nanocellulose is combined with starch, the peaks of both materials (starch and nanocellulose) can be seen, and compared to pure samples, changes in the location and intensity of these peaks can be observed, consistent with changes in the molecular structure or compounds in the example is mentioned. The addition of tea extract to starch-nanocellulose composites can lead to changes in the FTIR spectrum, including an increase or decrease in the intensity of peaks or the appearance of new peaks, which may be caused by chemical interactions between the compounds. The FTIR spectrum of pure starch exhibits characteristic peaks indicative of its chemical structure. The peak observed at approximately 3200–3600 cm −1 corresponds to the stretching vibrations of hydroxyl (OH) groups, which are abundant in starch molecules 9 , 76 . Another prominent peak around 2900–3000 cm −1 signifies the stretching vibrations of C–H stretching of the glucose units 77 . Additionally, a peak at about 1640–1660 cm −1 suggests the presence of bound water molecules, reflecting the hydrated nature of starch. Moreover, a peak in the region of 1020–1150 cm −1 is associated with the stretching vibrations of C–O–C glycosidic linkages, which are fundamental to the starch molecular structure. The FTIR spectrum of pure nanocellulose reveals characteristic peaks that reflect its cellulose-based composition. A strong peak at around 3300–3600 cm −1 corresponds to the stretching of hydroxyl (OH) groups abundant in cellulose. Peaks in the range of 2900–2905 cm −1 are attributed to the C–H stretching vibration within the cellulose backbone 78 . Additionally, peaks around 1150–1100 cm −1 indicate the presence of a C–O–C glycosidic bond and a C–C ring, which are key structural elements of cellulose molecules 79 . In the FTIR spectrum of the starch-nanocellulose composite film, characteristic peaks of both starch and nanocellulose components are typically observed. These peaks overlap, indicating that both materials were successfully incorporated into the composite matrix. The interpretation of the peaks is consistent with those observed in the spectra of pure starch and nanocellulose, confirming the presence of the respective functional groups in the composite film. When tea extract is incorporated into the starch-nanocellulose composite film, additional peaks may appear in the FTIR spectrum, reflecting the presence of compounds from the tea extract. In FTIR spectroscopy, the peak counts for bands associated with polyphenols, flavonoids and other phytochemicals present in the tea extract in starch films may vary depending on the specific functional groups in the compounds. These peaks could include bands associated with polyphenols, polysaccharides, and caffeine (theine) present in the tea extract 80 . This finding is consistent with the literature reported 10 , 38 , 47 . Polyphenols typically show peaks associated with OH groups around 3300–3500 cm −1 , aromatic ring stretching around 1600–1700 cm −1 and C–O stretching around 1000–1300 cm −1 . Flavonoids show peaks for C=C stretching in the aromatic ring around 1600–1700 cm −1 and C–O stretching around 1000–1300 cm −1 (Fig.  7 a). The FTIR spectra of the different films after biodegradation under soil burial conditions showed different changes in the functional groups (Fig.  7 b). The FTIR spectra of various films after biodegradation under soil burial condition showed different functional group changes. Imam et al. 81 reported a decrease in OH absorbance (3100–3500 cm −1 ) due to starch degradation in starch-PVA-lignocellulosic fiber films after burial in soil for 120 days.

FTIR spectra of films ( a ) before and ( b ) after biodegradation under soil burial conditions (30 days).

EDX analysis

EDX analysis is a technique that can be used to analyze the elemental composition of a material. The main elements of the different films before and after biodegradation under burial conditions are shown in Fig.  8 . The main composition consisted of C, N, O, Na, P, and Ca. These results clearly show that the carbon content in the sample decreased after the biodegradation process. The EDX analysis revealed a reduction in carbon content and an increase in oxygen content in the starch film sample that underwent degradation in the soil. This result may be caused by the processes of decomposition and destruction of starch by microorganisms and other biochemical processes in the soil. These processes can lead to ion exchange between carbon and oxygen ions with other ions and consequently to a decrease in carbon content and an increase in oxygen in the samples. In addition, bacteria employ low molecular weight polymer chains in the process of microbial digestion. These intermediates serve as energy sources for the microbes and undergo breakdown into compounds including water, CO 2 , and other metabolic biomass 55 , 72 . Similar EDX results have been obtained in earlier reports 82 , 83 , 84 , 85 .

EDX spectrum of different films before and after biodegradation under soil burial conditions (30 days). ( a ) control (pure starch), ( b ) starch-2% nanocellulose, ( c ) starch-4% nanocellulose, ( d ) starch-6% nanocellulose, ( e ), starch-2% nanocellulose-tea extract, ( f ), starch-4% nanocellulose-tea extract, and ( g ) starch-6% nanocellulose-tea extract.

Effect of biodegradation process on soil properties

The ANOVA indicated a significant treatment (film type) and biodegradation time effect on the physical and chemical properties of soil under biodegradation ( p  < 0.01) (Table 3 ). In some treatments, such as moisture content, TOC, and pH, the interaction effect (treatment × time) of variable factors was not significant ( p  > 0.05).

Moisture content and water holding capacity (WHC)

The results showed that the presence of films in the soil increased soil moisture content and WHC compared to soil without films (p < 0.05) (Fig.  9 ). The capacity of soil to retain water is crucial for plant development since water is essential for the synthesis of glucose in plants 86 . The increase in moisture content of soils with buried films compared to control soils can be attributed to several factors related to the degradation of the film material and its effects on soil properties. Nanocellulose usually demonstrates pronounced hydrophilicity as a result of the many hydroxyl groups (OH) or hydrophilic functional groups that are included during the manufacturing process 87 . This strong hydrophilic property causes more moisture absorption and increases soil moisture content and WHC. When biodegradable films are placed in soil, they create a small environment that helps to retain moisture. This occurs by reducing evaporation and enhancing water holding. The film material acts as a barrier that limits water loss from the soil surface. This leads to higher moisture levels in the area surrounding the buried samples. As the film material decomposes, it releases organic matter into the soil, which helps to improve soil structure and porosity. This ultimately leads to an increase in the soil's ability to retain water. Additionally, the breakdown of the film material stimulates microbial activity in the soil, which can further impact soil moisture levels. Therefore, the observed increase in soil moisture levels is a result of reduced evaporation, improved soil structure, and increased microbial activity caused by the decomposition of the film material. Earlier reports showed that the starch-based films can enhance the soil's water holding capacity or moisture content. For instance, Nassaj-Bokharaei, et al. 86 found that the reinforced starch-based hydrogels with natural char nanoparticles, significantly improved soil WHC, nutritional indices (16–29% increase), and tomato plant growth (22–45% increase). Similarly, Sen and Das 88 reported that biodegradable films made from starch and polyvinyl alcohol (PVA) increased soil WHC (27.5%), improving soil quality and supporting plant growth conditions.

Effect of biodegradation process on ( a ) soil moisture content and ( b ) water holding capacity.

The highest microbial respiration was observed on the 10th day after the films were applied to the soil. This was related to the treatment of pure starch (188.53 mg CO 2 –C g −1 soil), which was to be expected. SNBTE films had lower soil respiration compared to starch-nanocellulose films within 10–30 days after addition to the soil, regardless of their concentration (Fig.  10 ). This phenomenon arises from the fact that it serves as a more readily available carbon source for soil microorganisms, leading to the production of a diverse array of starch-degrading enzymes 9 . Compared to pure starch films, starch-nanocellulose films can hinder the access of microbes to food sources and suitable conditions for their growth below the soil surface, leading to a decrease in microbial activity below the soil surface. Antibacterial and antifungal effects of active ingredients in black tea extracts can reduce microbial activity 43 , 44 . In addition, the physical and structural properties of films containing nanocellulose, compared to pure starch films, can reduce the permeability to air and moisture, which in turn limits the access of microbes to food sources and suitable conditions for their growth below the soil surface. This can lead to a decrease in microbial activity below the soil surface. Soil respiration rates in soils containing buried film samples are higher than those in control soils due to several interrelated factors. Biodegradable films in soil become a substrate for microbial colonization and activity, leading to an increase in soil respiration rates as the microorganisms degrade the organic components of the film material. The presence of film material also stimulates the growth and activity of microbial biomass, which further increases soil respiration rates. In addition, the degradation of the film material leads to changes in the physicochemical properties of the soil, which can influence the microbial respiration processes. This increase in soil respiration is the result of the combined effects of increased substrate availability, microbial biomass growth, and altered soil conditions. This fact is well supported by the finding of Malekzadeh et al. 9 and Nassaj-Bokharaei et al. 86 .

Effect of biodegradation process on soil respiration.

The data presented in Table 4 show the biodegradation of the different film types under soil burial conditions at different time intervals (10, 20, and 30 days). In terms of CWEOC, the data show that the control group (pure starch) has higher values than soil alone across all time points. Films with nanocellulose, both alone and with tea extract, generally have lower CWEOC values than the control group, suggesting a potential reduction in the leaching of organic carbon into the soil by these film compositions. Interestingly, the addition of tea extract appears to have a slightly decreasing effect on CWEOC values, which is particularly noticeable for the 4 and 6% nanocellulose compositions. The HWEOC values generally follow a similar trend, with the control group showing consistently higher values compared to the pure soil. Films containing nanocellulose generally had lower HWEOC values compared to the control group, indicating a potential reduction in the release of water-soluble organic carbon into the soil by these film compositions. Again, the addition of tea extract appears to have a slightly decreasing effect on HWEOC content, particularly pronounced with the 2% nanocellulose composition. Analysis of the TOC content shows a similar pattern, with the control group consistently showing higher values than the soil alone. Films containing nanocellulose generally have higher TOC values than the control group, indicating a possible increase in the total organic carbon content in the soil due to the decomposition of these films. The addition of tea extract generally shows a mixed effect on TOC content, with effects varying depending on nanocellulose concentration.

The increase in CWEOC and HWEOC of soils containing buried starch/nanocellulose film samples compared to control soils can be explained by several factors related to the degradation of the film material and its subsequent interaction with the soil environment. Starch-nanocellulose films undergo biodegradation processes after burial in the soil, which are promoted by microorganisms in the soil. The presence of starch in films can increase microbial activity and diversity in the soil, as these materials can serve as substrates for the growth of soil microflora 88 . As these films decompose, organic carbon compounds are released into the surrounding soil matrix. Starch, a polysaccharide, is enzymatically hydrolyzed by soil microbes, resulting in the release of soluble sugars and other organic compounds. Similarly, nanocellulose, a carbohydrate-based material, also contributes to the organic carbon pool during decomposition. The presence of these organic compounds increases the CWEOC and HWEOC content in the soil. In addition, the degradation of starch-nanocellulose films can stimulate microbial activity, leading to an increase in microbial biomass and metabolic processes, which in turn contribute to the release of organic carbon into the soil solution. In addition, the physical structure of the film material can promote soil aggregation and improve soil porosity, which facilitates the penetration of water and organic carbon compounds, thereby increasing the CWEOC and HWEOC content. The observed increase in CWEOC and HWEOC content in soils containing buried starch-nanocellulose film samples compared to control soils is therefore a consequence of the complex interactions between the degrading film material, soil microorganisms and soil organic carbon dynamics. In a similar study, Sen and Das 88 reported that after 35 days of burying starch-based antibacterial films in the soil, the amount of carbon increased by 14.4% compared to the control treatment. Li et al. 89 in the study on the biodegradability of starch, polylactic acid and cellulose-based mulches on soil quality, found that these mulches increase the amount of organic carbon in the soil, but their changes are very small and depend on the production system and incubation time.

Figure  11 shows the total nitrogen content in the soil samples and the different films at different time intervals (10, 20, and 30 days) after biodegradation under soil burial conditions. Considering solely the total nitrogen content in the soil, it appears to remain relatively stable over time, indicating minimal variations in nitrogen levels under the specified conditions. In contrast, the total nitrogen content in the control group (pure starch) and in the different films generally increases over time, indicating a release or accumulation of nitrogen during the biodegradation process.

Effect of biodegradation process on soil total nitrogen.

Comparing the different film compositions, those containing nanocellulose showed a higher total nitrogen content than the control group at all time points, indicating a possible contribution of nanocellulose to nitrogen availability in the soil during biodegradation. Interestingly, the addition of tea extract along with nanocellulose appears to have a mitigating effect on total nitrogen content, with lower values observed compared to nanocellulose-only compositions, particularly noticeable at the 4% and 6% nanocellulose concentrations. The increase in total nitrogen content of soils containing buried starch-nanocellulose film samples compared to control soils can be attributed to several mechanisms related to the decomposition of the film material and its interactions with the soil ecosystem. Starch nanocellulose films, when buried in the soil, are degraded by microorganisms in the soil. During this process, the organic components of the film material, including starch and cellulose, are broken down into simpler compounds, some of which contain nitrogen, such as amino acids and proteins. The decomposition of the film material releases nitrogen into the soil matrix, which contributes to an increase in the total nitrogen content. In addition, the presence of starch and nanocellulose in the film material can increase microbial activity in the soil, leading to increased nitrogen mineralization and turnover of organic nitrogen compounds. In addition, the degradation of the film material can change the physical properties of the soil, such as porosity and water retention, which can affect nitrogen availability and cycling processes. The increase in total nitrogen observed in soils with buried starch nanocellulose film samples compared to control soils is therefore a consequence of interactions between the decomposing film material, soil microorganisms and nitrogen dynamics in the soil. Sen and Das 88 reported that the amount of available nitrogen and total nitrogen in the soil containing starch-based antibacterial films was 86% and 157% higher than the control, respectively. In another study, Zhao et al. 90 reported that the biodegradable films enhanced soil nitrogen levels but decreased organic carbon content, affecting the C/N ratio. Koskei et al. 91 found that biodegradable residual films in agriculture improved soil nitrogen content and reduced the C/N ratio.

Electrical conductivity (EC) and pH

Figure  12 shows the effects of biodegradation of the film on the electrical conductivity (EC) and pH of the soil over 30 days. As for pH, slight variations were observed between the different film compositions and over time. The control group and the film compositions containing tea extract tended to have slightly lower pH values than the soil alone and the other film compositions, indicating a potential acidifying effect. Conversely, film compositions with higher concentrations of nanocellulose show pH values closer to those of the soil alone, indicating less influence on soil acidity. The decrease in pH might have been caused by the decomposition of the buried film's organic matter by soil microorganisms, which produce organic acids 88 . In addition, the degradation of starch-nanocellulose films can also influence soil pH dynamics. Initially, the decomposition of organic matter from the film material can release acidic compounds into the soil, resulting in a temporary decrease in pH. However, as decomposition progresses, the release of organic acids can be balanced by the accumulation of alkaline substances such as carbonates and bicarbonates produced by microbial activity. This shift towards alkalinity can contribute to an increase in soil pH over time.

Effect of biodegradation process on ( a ) EC and ( b ) pH.

When analyzing the EC values, fluctuations can be observed over time and with the different film compositions. In general, the control group (pure starch) and the film compositions with tea extract tend to have higher EC values than the soil alone and the other film compositions. Conversely, films with higher concentrations of nanocellulose (4% and 6%) have lower EC values, indicating a potential mitigating effect on soil salinity. The increase in EC of soils containing buried starch nanocellulose film samples compared to control soils can be attributed to several interrelated mechanisms related to the decomposition of the film material and its interaction with the soil environment. Starch nanocellulose films, when buried in soil, undergo microbial degradation, which is promoted by soil microorganisms. During this process, the organic components of the film, such as starch and cellulose, are broken down into simpler compounds, releasing ions and organic matter into the soil matrix. This organic matter contributes to the soil's nutrient pool and can increase microbial activity, leading to increased mineralization of organic compounds and subsequent release of ions, including those that contribute to EC, such as potassium, calcium and magnesium. Sen and Das 88 reported a 10.5% decrease in soil pH as a result of buried starch-based antibacterial films. Qi et al. 92 reported that after 4 months, soil pH values ​​decreased in treatments containing 1% bio-macroplastic compared to the control. But soil EC has been increasing. Li et al. 89 reported substantial changes in soil EC and pH, suggesting that cellulose and starch-based mulch films significantly impact these soil properties.

MDW and GMD

The provided data in Table 5 presents the effect of the biodegradation process on MDW and GMD after 30 days. The higher the MWD and GMD values, the better the water-stability of the soil aggregate. On the other hand, the lower the values, the worse the water-stability 90 . Analyzing MDW, the control group (pure starch) exhibits an MDW value of 0.035 mm, slightly higher than that of the soil alone, which has an MDW of 0.024 mm. The film compositions containing nanocellulose, either alone or with tea extract, generally show slightly lower MDW values compared to the control group, indicating a potential influence of these film compositions on the aggregation of soil particles. However, the differences are relatively small, suggesting that the addition of nanocellulose and tea extract may have a limited impact on the stability and size of soil aggregates. Examining GMD, similar trends are observed, with the control group displaying a GMD of 1.130 mm, slightly higher than the soil alone, which has a GMD of 1.096 mm.

Film compositions containing nanocellulose, both alone and with tea extract, generally exhibit GMD values comparable to or slightly higher than the control group, indicating a potential influence of these compositions on the overall size distribution of soil aggregates. However, once again, the differences are relatively minor, suggesting that the addition of nanocellulose and tea extract may have a limited effect on the geometric mean diameter of soil aggregates. A possible explanation for the increase in the MDW and GMD after the biodegradation of SNBTE films can be attributed to the physical and chemical effects of these substances in the decomposition process and the changes they cause in the soil structure. The presence of nanocellulose and tea extract in the films likely leads to an increase in the density and stability of soil aggregates, because these materials can act as adhesion agents and bind soil particles together, which can lead to the creation of larger and more stable soil aggregates that they are created by the decomposition of films. In previous studies, microplastics were also shown to reduce macroaggregate fractions and alter water-stable aggregate profiles 93 . Zhao et al. 90 reported that films with biodegradable and plastic residues applied at high levels reduced MWD and GMD. This means that the films prevent the formation of macro-soil aggregates (> 0.25 mm). The difference of this result compared to the results obtained in this research can be caused by the origin of the film used.

Policy implications

The investigation into the biodegradation behavior of SNBTE films under soil burial conditions and their impact on soil physical and chemical properties suggests several policy implications. Firstly, there's a need for comprehensive environmental impact assessments to evaluate the suitability of these biodegradable films in agriculture, considering their interaction with soil and potential effects on soil quality. Regulatory frameworks should be established or updated to ensure the responsible use of biodegradable materials, incorporating standards for film composition and degradation rate to protect soil health. Governments and agricultural agencies can encourage the adoption of sustainable agriculture practices by promoting the use of biodegradable films, thus reducing plastic pollution and preserving soil fertility. Additionally, increased funding for research and development can drive innovation in biodegradable film technology, leading to more environmentally friendly alternatives. Education and awareness campaigns are essential to inform stakeholders about the benefits and proper application of biodegradable films, fostering a more sustainable agricultural sector.

Limitations, challenges, and prospects of research

The study of the biodegradation behavior of SNBTE films under soil burial conditions provides both challenges and opportunities for the development of sustainable packaging materials. By replacing conventional plastic films, this film system may offer a new and biodegradable material for food packaging and other applications. However, there are several limitations and challenges in the study of biodegradable packaging materials. Firstly, the biodegradation process is complex, and many factors can influence it, including soil composition, potential microbial activity, and environmental conditions. Accurately assessing the kinetics and mechanisms of biodegradation of composite films is therefore a big challenge. Secondly, the biodegradation rates of various components in the composite films can be different. Particularly for nanocellulose, the biodegradation mechanism of this component is not clear, which may change the biodegradability of the overall film, as well as the dynamics of nutrient release. In addition, the potential impact of the degradation by-products of composite films on soil respiration and nutrient availability also requires careful evaluation to verify the compatibility of the materials with the surrounding environment. In conclusion, this study takes a step towards the development of sustainable packaging materials by exploring the biodegradation behavior of unique composite films. Additionally, our work provides strategies to optimize the biodegradable behavior of composite films and further enhance their performance in environmental applications. The knowledge gained in this study is crucial for the development of formulations that are not only biodegradable but also have a low environmental impact. This will help to move the packaging industry one step closer to a more sustainable path. However, the development and evaluation of biodegradable composite films also require close collaboration between material scientists, microbiologists, and environmental engineers. Interdisciplinary cooperation is essential for the sustainable development of biodegradable composite films. The problem-solving process in this area requires considerable support from all areas.

Conclusions

The study concludes that SNBTE films biodegrade effectively under soil burial conditions, demonstrating their potential as an environmentally friendly alternative to conventional plastics. The incorporation of 6.0% nanocellulose managed to improve the tensile strength up to 10.54 MPa by 40.4% compared to pure starch film. The addition of tea extract to the films significantly decreased their biodegradation rate, which is due to the polyphenolic compounds that are likely to stimulate microbial activity. The degradation of these films had a positive effect on soil health by increasing microbial activity and organic matter content, while pH and EC remained stable. These results underline the dual benefit of using SNBTE films, i.e. reducing plastic waste and improving soil quality. Therefore, these biodegradable films are promising for use in sustainable packaging and agriculture, contributing to both waste management and improved soil health.

Data availability

The data provided in this study is available with the corresponding author and can be presented on considerable request.

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Malekzadeh, E., Tatari, A. & Dehghani Firouzabadi, M. Effects of biodegradation of starch-nanocellulose films incorporated with black tea extract on soil quality. Sci Rep 14 , 18817 (2024). https://doi.org/10.1038/s41598-024-69841-2

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