Sweet without Sugar: The Secret of "Sweet Tea" Revealed

When people hear "sweet tea," many might think of iced tea with plenty of sugar added.

However, the "sweet tea" studied here is not a typical tea with sugar added. It refers to Lithocarpus litseifolius, a plant from the beech family that grows in certain regions of China and has traditionally been used as a naturally sweet tea.

The drink made from the young leaves and buds of this plant is sweet without any added sugar. This is due to the large accumulation of a plant compound called "dihydrochalcone" in the leaves.

The main components are phloridzin and trilobatin. These are flavonoid compounds bound with sugar, thought to be involved in plant defense and environmental adaptation, while being noted for their strong sweetness as natural ingredients for humans.

However, how sweet tea produces such a large amount of sweet compounds through chemical reactions was not well understood for a long time.

A research team, including the Tea Research Institute of Zhejiang University, has now examined this complex "sweetener factory within the plant" at the genetic and enzymatic levels.

30% of the Dry Weight of Young Leaves is Sweetness-related Compounds

The most notable feature of sweet tea is its extremely high content of dihydrochalcones.

According to the original research paper, the proportion of phloridzin and trilobatin in the leaves can reach about 21-33% of the dry weight. This means that a significant portion of the young leaves, excluding moisture, is composed of sweetness-related compounds.[1][2]

Phloridzin is also found in apple leaves and bark, but the content in sweet tea is higher than in apple plants. Thus, this plant is suitable not only as a source of natural sweet compounds but also as a model plant for studying the biosynthesis of dihydrochalcones.

In recent years, the food industry has sought technologies to reduce sugar usage while maintaining taste.

However, sugar is not just an ingredient for sweetness. In beverages, it affects mouthfeel and aroma perception, and in confectionery, it influences volume, browning, moisture retention, and texture. Therefore, simply replacing sugar with another sweetener often fails to replicate the original taste and quality.

Still, if plant compounds that provide sufficient sweetness in small amounts can be produced stably, they could offer a new option for reducing sugar in beverages, dairy products, confectionery, health foods, and more.

The significant aspect of this research is that it revealed the "blueprint" used by the plant to produce sweet compounds, rather than a method for mass harvesting the raw material.

Explaining the Previously Unexplained "Double Bond" Processing

When plants produce dihydrochalcones, multiple raw materials and enzymes react sequentially.

To ultimately produce phloridzin and trilobatin, the basic structure "phloretin" must first be synthesized. This process requires a reduction reaction that converts double bonds between carbon atoms in the starting material into single bonds.

Previously, it was believed that a double bond reductase directly acted on a substance called p-coumaroyl CoA, which is bound to coenzyme A.

However, past experiments using apples reported cases where the candidate enzymes did not work as expected. Reactions were not confirmed in test tubes, or the desired compounds were not sufficiently produced even when genes were introduced into other organisms, leading to inconsistent results.

The research team hypothesized that "the stage at which the reduction reaction occurs might be different."

Investigations revealed that the reduction of the double bond occurs not in the CoA-bound form as previously assumed, but in an aldehyde form produced along the way.

First, an enzyme named LlCCR converts p-coumaroyl CoA into p-coumaraldehyde. Then, an enzyme called LlDBR1 reduces the double bond of the molecule in its aldehyde state, producing dihydro-p-coumaraldehyde.

This discovery may explain why some enzymes in past studies did not react with CoA-bound substances.

In other words, researchers were trying to make "workers in a different process" do the job. In fact, LlDBR1 was responsible for the subsequent aldehyde process.

Examining 24 Candidate Genes One by One

The research team selected 24 genes from sweet tea's gene expression information that might be related to the synthesis of sweet compounds.

The targets included cinnamoyl CoA reductase, double bond reductase, aldehyde dehydrogenase, 4-coumaroyl CoA ligase, chalcone synthase, and glycosyltransferase.

In the study, candidate genes were expressed in E. coli to produce enzymes, and their reactions with substances were confirmed in test tubes. They not only examined the presence of genes but also verified reaction rates and substrate selectivity.

Furthermore, they used antisense oligonucleotides to temporarily suppress specific gene functions in plants and observed changes in dihydrochalcone content in the leaves.

Suppressing genes like LlCCR, LlDBR1, LlALDH1, and Ll4CL2 resulted in a decrease in the target sweet compounds. Similarly, suppressing LlP4′GT, which produces trilobatin, or LlP2′GT1, which produces phloridzin, also reduced the respective compound levels.

Both the enzyme experiments in test tubes and the gene suppression experiments in living plants pointed in the same direction, strongly supporting the involvement of each enzyme in biosynthesis within the actual plant.

The Path to Phloretin Wasn't Just One

Another important discovery by the research team was that there was more than one pathway to the basic structure of sweet compounds, phloretin.

It was shown that phloretin could be produced from the common intermediate dihydro-p-coumaraldehyde via at least two routes.

In the first route, LlALDH1 converts the aldehyde to an acid, and Ll4CL2 adds CoA again. Then, chalcone synthase LlCHS1 reacts to produce phloretin.

In the second route, LlCCR is involved in a reverse reaction, supplying the necessary substances for phloretin synthesis from the intermediate. LlCHS1 then acts to produce phloretin.

It's like having two roads leading to the same destination.

Even if raw materials or enzymes are lacking in one pathway, production can continue by utilizing the other. For plants, this means they can adjust metabolic flows according to growth conditions and environmental factors.

From the perspective of the food industry, it may be possible to choose the more efficient production route between the two. When producing phloretin or trilobatin in fermentation tanks using yeast or E. coli, it might be possible to design a route suitable for the host microorganism.

Enzyme That Hardens Wood Also Involved in Sweet Compounds

Particularly intriguing is the multifunctionality of LlCCR.

Enzymes called CCR are typically involved in the synthesis of lignin, which strengthens plant cell walls. Lignin is a crucial component that hardens plant stems and trunks and supports water-conducting tissues.

However, in sweet tea, LlCCR was involved not only in lignin synthesis but also in the production of the sweet compound dihydrochalcone.

The carbon resources available to plants are not unlimited. They need to choose whether to allocate the same starting materials to lignin, which strengthens the body, or to dihydrochalcone.

LlCCR is thought to act as a "switch" that regulates the flow of metabolites near this branching point.

In young, soft leaves, there is no need to produce large amounts of lignin yet, while secondary metabolites are needed for protection against insects and ultraviolet rays. Although the exact defensive function of dihydrochalcone within the plant remains a future challenge, the concentration of sweet compounds in young leaves may be related to the plant's growth strategy.

Why Are Young Leaves Sweeter?

The study also investigated why dihydrochalcones accumulate abundantly in young leaves.

One reason is that LlCCR can efficiently process the starting materials. Another reason is that glycosyltransferases downstream in the synthesis pathway are strongly expressed in young leaves.

The position where sugar is added to phloretin changes the final compound.

When LlP4′GT acts, trilobatin is mainly produced, and when LlP2′GT1 acts, phloridzin is mainly produced. Because these glycosyltransferases work actively, the produced phloretin is rapidly converted into stable glycosides and accumulates in the leaves.

The reaction of adding sugar is not just a finishing touch for plants. It is an important process that changes solubility in water, movement within cells, toxicity, and ease of storage.

In other words, sweet tea excels not only in its ability to produce sweet molecules but also in its ability to process and store them in a stable form.

Is the Sweetness 300 Times That of Sugar Really True?

A recent review on functional sweet tea in China introduced literature information stating that dihydrochalcone compounds like trilobatin and phloridzin have sweetness approximately 300 times that of sucrose.[3]

However, the figure "300 times sweeter" does not simply mean that using 1/300th of sugar will produce the same product.

High-intensity sweeteners differ from sugar in the time it takes to perceive sweetness and the duration of sweetness in the mouth. Reports suggest that phloridzin and related compounds have a slow onset of sweetness, with a refreshing aftertaste appearing later.

Additionally, the combination with bitter compounds like catechins in foods may weaken the perception of sweetness. On the other hand, when combined with a small amount of sugar, the sweetness may synergistically increase.

Therefore, in actual product development, it is more practical to combine a small amount of sugar with other sweeteners, flavors, and acidity rather than replacing all sugar with one type of sweet compound.

The important thing is not just the intensity of sweetness. "Sweetness time design" that includes the sweetness at the moment it enters the mouth, the aftertaste after swallowing, and harmony with bitterness, astringency, and aroma is necessary.

The Era of Microorganisms Producing Natural Sweet Compounds

Once the biosynthetic pathway is clarified, production methods other than extracting components from plants can be considered.

For example, introducing necessary genes into yeast or E. coli to ferment and produce the target compounds from sugars or plant-derived raw materials.

In microbial production of phloretin so far, the reduction step of double bonds has often been a bottleneck. Therefore, methods of adding intermediate raw materials externally or borrowing reduction enzymes from non-plant sources have been used.

By utilizing the combination of LlCCR and LlDBR1 discovered this time, it may be possible to construct a reaction system closer to the plant's original pathway.

Furthermore, after producing phloretin, activating LlP4′GT can produce more trilobatin, and activating LlP2′GT1 can produce more phloridzin, allowing for adjustment of the production.

However, synthesizing at the laboratory level and producing cheaply and in large quantities as food are separate issues.

Many challenges need to be addressed, such as enzyme stability, production speed, the amount of by-products, purification costs, fermentation raw materials, wastewater treatment, food standards, and changes during storage.

Nonetheless, identifying the two biosynthetic routes and key enzymes means that developers have moved from a stage of trial and error in the dark to a stage where they can make improvements while looking at the blueprint.

Potential for Use in Breeding

The results of this study may be applicable not only to microbial fermentation but also to the breeding of sweet tea itself.

By selecting individuals with strong functions of LlCCR, LlDBR1, LlP4′GT, and LlP2′GT1, it may be possible to cultivate varieties with higher sweet compound content.

It can also help optimize the harvest timing. Even if it's known that young leaves have more compounds, harvesting too early may reduce yield, while harvesting too late may decrease sweet compound content.

By combining leaf growth stages, gene expression, compound content, and yield, the most efficient harvest timing can be determined.

On the other hand, excessively increasing only the sweet compounds may affect plant growth and disease resistance. If lignin and dihydrochalcone compete for the same raw materials, directing too much metabolism towards sweet compounds could weaken branches and stems.

In breeding, a balance that includes not only sweetness but also yield, disease resistance, cold resistance, and ease of cultivation is necessary.

"Naturally Derived" Does Not Guarantee Health Benefits

It is important to note that this study is not a clinical study verifying weight loss or diabetes prevention in humans.

The focus of the research is on the pathways through which sweet compounds are produced within the plant and the identification of the involved enzymes.

For phloridzin and trilobatin, effects related to sugar metabolism, antioxidation, and inflammation have been reported in cell and animal experiments. However, these results cannot be directly applied to everyday human consumption.

How the ingested compounds are broken down in the digestive tract, how much is absorbed, and what effects occur with long-term consumption need to be confirmed in separate studies.

Moreover, the fact that something is "derived from plants" or "naturally derived" does not guarantee safety or health benefits.

To use it as food, purity, impurities, intake amount, metabolites, allergies, and interactions with drugs must be evaluated. In some countries and regions, it may also be necessary to undergo examination as a new food material or food additive.

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