As a formulation developer at a nutrition company, you may need a clear answer before adding an HMO to infant formula, a nutrition powder, a drink, yogurt, or a bar: will the product keep the intended HMO amount after processing and through the end of shelf life?
Tests on specific HMO ingredients have shown that many can remain stable during pasteurization, ultra-high-temperature (UHT) processing, fermentation, drying, baking, and storage. Still, "heat stable" alone does not tell you whether a commercial formula will pass. The answer depends on the exact HMO ingredient, amount, product acidity (pH), temperature, holding time, moisture, other ingredients, package, and storage period.
We manufacture 2'-FL, 3-FL, LNnT, LNT, 3'-SL, and 6'-SL. Existing studies and regulatory files can help your team choose conditions for a first trial. Some of the six HMO types have more finished-product data than others.
I would not approve an HMO for a commercial formula based only on a general heat-stability statement. Ask for the conditions behind the statement, then measure the supplied HMO in your real formula before processing, after the step most likely to affect it, and during shelf life.
Do not ask only, “Are HMOs heat stable?” Ask, “How much of this HMO remains in this formula after this process and at the end of shelf life?”
Define the Question First
Decide What Stable Means for Your Product
A product can retain the target HMO level and still develop other problems. A powder may absorb moisture and cake. A drink may retain the HMO while its color or flavor changes or sediment forms during storage.
Set clear acceptance limits for HMO identity and content. Also define acceptable moisture, texture, microbiological quality, flavor, and appearance through the end of shelf life. A single "heat stable" statement cannot replace those checks.
You will also see two technical terms in stability files. Hydrolysis means that water, pH, and heat split part of the HMO into smaller sugars. Isomerization means that the atoms rearrange into a related molecule. Either change can reduce the amount of the named HMO that the laboratory measures.
Temperature
Record the highest temperature together with the hold time. A few seconds of UHT treatment is not equivalent to keeping a product warm for days or weeks.
Time
Include heating, holding, cooling, fermentation, and shelf life. A moderate temperature applied for a long time may change an HMO more than a higher temperature applied for seconds.
pH
Acidic or alkaline conditions may split or rearrange an HMO, especially during long heating or storage. Test the pH of the finished product rather than water alone.
Moisture
Water activity measures how freely water can move in a product. It can affect powder caking, flow, chemical change, and microbial growth.
Finished Formula
Proteins, minerals, sugars, live cultures, and other ingredients can change how an HMO behaves. Test the commercial formula, not only an HMO solution.
Measure the Named HMO, Not Only Total Oligosaccharides
An HMO assay tells you how much of a specific HMO remains. It shows whether the result meets the limit you set for the trial. It does not prove a health benefit, and results from a different formula may not apply to yours.
The test method must separate the target HMO from lactose, related carbohydrates, and other HMOs. The AOAC First Action 2022.07 study describes one method for measuring seven named HMOs in infant formula and adult nutrition products.
Start with Existing Results
What Existing Tests Show for Six HMOs
Use the table to identify useful starting conditions for your trial. Each result applies only to the tested HMO ingredient, formula, process, test method, and storage period. It does not guarantee the same result for every HMO ingredient or finished product.
| HMO | Tested Products or Conditions | What Happened | Source |
|---|---|---|---|
| 2'-FL | Whole milk, yogurt, UHT milk, infant formula, and a 2'-FL/DFL powder | 2'-FL stayed within the reported ranges under the tested dairy processes and storage periods. Long stress tests also found breakdown under acidic conditions and molecular rearrangement near neutral or alkaline pH. | 2'-FL and 3-FL finished-product study |
| 3-FL | Milk, yogurt, UHT milk, infant formula, cereal bars, juice, and milkshakes | 3-FL stayed within the reported ranges in several tested products, including a heated cereal bar and pasteurized or UHT liquids. | EFSA 3-FL safety assessment |
| LNnT | Infant formula, yogurt, citrus drink, and pasteurized or UHT chocolate milk | LNnT remained within the reported ranges through the tested processes and storage periods, including an infant-formula study that ran for up to 900 days. | FDA GRN 1067 notice |
| LNT | Infant formula and bulk powder; discussion of other foods partly uses LNnT data | LNT remained within the reported ranges in the tested infant formula and stored powder. Some discussion of other foods relies on LNnT data rather than direct LNT tests. | FDA GRN 1068 notice |
| 3'-SL | Infant formula, milk, yogurt, powder, and aqueous pH stress tests | 3'-SL remained within the reported targets in infant formula and milk. It also stayed within target for 45 days in refrigerated yogurt, but not after 15 days in yogurt stored at room temperature. | FDA GRN 1074 notice |
| 6'-SL | Infant formula, bulk powder, and aqueous pH stress tests | 6'-SL remained within the reported range in the tested infant formula. Some discussion of additional foods relies on 3'-SL and other related-HMO data rather than direct 6'-SL tests. | FDA GRN 881 notice |
Direct finished-product tests cover 2'-FL, 3-FL, and LNnT across several product types. LNT has direct infant-formula data, but some discussions of other foods use LNnT data. Although LNT and LNnT share a molecular formula, their structures differ. 6'-SL has direct infant-formula and stress-test data, while some discussions of additional foods use results from 3'-SL or related HMOs.
Two suppliers may sell ingredients under the same short HMO name, but their production strains, purification processes, and specifications can differ. Use stability data from another manufacturer to plan a trial, but test your actual ingredient before approving it.
Data from a related HMO can show you where problems might occur and when to take samples. You still need to test the HMO in the formula your company plans to sell.
Thermal Processing
Heat Exposure Means Temperature Plus Time
Your process always combines temperature with time. Pasteurization usually applies moderate heat for a short hold. UHT processing applies a higher temperature for a much shorter time. During baking, the surface and center of the product also reach different temperatures and moisture levels. One generic heat-resistance number cannot describe all three processes.
Our 2'-FL Stayed Stable in a Three-Batch Heat Test
We tested three production batches of our 2'-FL as a 0.15% aqueous solution under two processing simulations. We compared each heated sample with its starting measurement to check whether short heat exposure reduced the measured 2'-FL content.
88-95°C for 5 Minutes
We held the solution within this temperature range for five minutes to simulate a milk-powder heat process.
140°C for 3 and 5 Minutes
We tested both a 3-minute hold and an extended 5-minute hold at 140°C to examine short exposure at a much higher temperature.
No Consistent Loss in Measured 2'-FL Content
Across the three batches, the assay results stayed close to the starting values. Neither heat condition caused a consistent reduction.
Nutrition companies can use this internal test to choose starting conditions for evaluating our 2'-FL in milk powder and other short heat processes. This test does not guarantee the same result in every finished product. Your formula may behave differently because pH, proteins, minerals, sugars, moisture, and the addition point all matter. Cooling, packaging, and storage matter too. Your team should test how much 2'-FL remains in your commercial formula after processing and storage.
Milk, Yogurt, and UHT Products
A peer-reviewed food-application study found that 2'-FL and 3-FL stayed within the reported ranges in whole milk, yogurt, and UHT milk. The researchers followed whole milk for 22 days and yogurt for 36 days at 5°C, and UHT milk for three months at room temperature.
The EFSA assessment for 3-FL reports stability results for pasteurized juice, pasteurized and UHT milkshakes, fruit yogurt, and cereal bars. These results can help your team plan a first trial in those categories, but you still need to test the HMO in your own formula.
A Decomposition Temperature Is Not a Processing Limit
A thermal-characterization study of crystalline and amorphous 2'-FL measured decomposition at about 210 to 212°C, around 10°C above lactose in the same laboratory test. This number does not mean that a nutrition company should process 2'-FL at 210°C. That laboratory test used an isolated ingredient, not a commercial formula with its own moisture level and processing time.
Moisture affected the powder well before decomposition. In the same study, more available water caused amorphous 2'-FL to become softer and more mobile at a lower temperature. This change can cause caking and poor powder flow even when the HMO amount remains within target.
Heat Can Also Change the Rest of the Formula
HMOs can take part in the Maillard reaction, which can cause browning when sugars react with proteins during heating or storage. A 2025 milk-protein model study found that 2'-FL and LNnT reacted at different rates with three milk proteins.
A model system cannot predict every infant formula or nutrition drink. It does show why your stability trial should check color, flavor, protein changes, and related carbohydrates as well as the HMO amount in a heated, protein-rich product.
Acid, Alkali, and Time
Review pH and Heat Together
Stress tests deliberately use harsher conditions than normal production so researchers can find possible problems faster. They show how an HMO may break down or rearrange, but they do not tell you exactly how much will remain in a commercial product. Four weeks at 80°C cannot predict the result of a short pasteurization step.
| Tested HMO | Test Conditions | What Happened | What to Do in Product Development | Source |
|---|---|---|---|---|
| 2'-FL/DFL mixture | Aqueous acidic or neutral systems stored at 60°C for 8 weeks or 80°C for 4 weeks | The mixture began to break down below pH 5 and rearrange above pH 6. A small amount of rearrangement also occurred near neutral pH. | Do not use a long heated-storage result to predict short pasteurization. In a heat-stress test, measure the target HMO and any related carbohydrates that form. | EFSA 2'-FL/DFL assessment |
| LNT | Aqueous pH 4.5 or 6.8 at 60°C for 8 weeks or 80°C for 4 weeks | Acidic conditions split part of the amorphous LNT into smaller sugars. Near neutral pH, a small amount changed into an LNT fructose isomer. | For an acidic product with a long hot hold, measure how much LNT remains. Also test the actual powder form and moisture condition. | FDA GRN 1068 notice |
| 3'-SL | Aqueous pH 3.0 to 9.0 at 35°C for 28 days, plus separate strong-acid and strong-base tests | 3'-SL remained stable near pH 6.9. A small amount broke down at pH 5.5, extensive breakdown occurred at pH 3, and rearrangement occurred at pH 9. | Acidic drinks and fermented products require a test at the actual pH, temperature, culture, and shelf life. | FDA GRN 1074 notice |
| 6'-SL | Aqueous pH 3.0 to 9.0 at 35°C for 28 days; powder at 80°C for 28 days under two humidity conditions | 6'-SL remained stable near neutral pH. About 3% broke down at pH 5, extensive breakdown occurred at pH 3, and higher humidity increased rearrangement in the powder. | Review pH and moisture together. A dry-powder result cannot predict how 6'-SL will perform in an acidic liquid. | FDA GRN 881 notice |
Acidic Products May Still Be Suitable
Strong acidity combined with long heating can damage some HMOs, but this does not mean every acidic food will fail. In the tests summarized above, 3-FL stayed within the reported range in a pasteurized juice drink for 28 days at 5°C, and LNnT remained within the reported range in a citrus drink after the process and storage period used in the test.
Test pH, temperature, and time together. An acidic drink with a brief heat step can perform very differently from an HMO solution held hot for weeks.
A Live Culture May Use the HMO during Storage
In one 3'-SL test, the HMO stayed within the target range for 45 days in refrigerated yogurt, but not after 15 days in yogurt stored at room temperature. Microorganisms may have consumed some of the 3'-SL during storage.
For yogurt, fermented drinks, and synbiotic products, measure the HMO during fermentation and storage. One culture may use a particular HMO more readily than another, so a heat-only test cannot answer the full question.
Shelf Life and Packaging
Storage and Packaging Can Create More Risk Than Processing
A product may spend only a few minutes in a heat exchanger or dryer, then remain in its package for months or years. Your shelf-life trial should cover temperature, humidity, light, oxygen, and the package barrier. If consumers will open the package repeatedly, include that use pattern in the trial.
Protect Low-Moisture Powders
Use a package with an effective moisture barrier and close containers promptly. Measure moisture and water activity because caking or poor flow can appear before a large change in the HMO amount.
Test the Final Package
Stability data for the bulk HMO ingredient cannot tell you how long the product will last in a sachet, tub, bottle, bar wrapper, or ready-to-drink package. Each package creates a different environment around the formula.
Some HMO powders have stayed within their reported ranges during multi-year room-temperature or accelerated-storage tests. Use those results to plan your test, but do not apply the longest published shelf life to an HMO from a different manufacturer or to a different finished package.
Accelerated storage raises temperature or humidity to reveal likely problems sooner. It can help compare formulas and packages. Before setting an expiration date, use real-time data or validate a model that links the accelerated test to normal storage.
Plan the Product Trial
Build the Test Around the Finished Product
Follow the product through its real manufacturing process and storage period. Take samples where heat, acid, moisture, live cultures, or packaging are most likely to change the HMO or the rest of the formula.
Dry Nutrition Powders and Infant Formula
Test after blending or spray drying and again during storage. Measure the named HMO, related carbohydrates, moisture, water activity, color, powder flow, and reconstitution.
Pasteurized, UHT, and Ready-to-Drink Products
Sample before heating, immediately after heating, after packaging, and during shelf life. Run the trial with the actual proteins, minerals, sugars, and pH.
Yogurt and Fermented Products
Measure the HMO during fermentation and refrigerated storage. A live culture may consume one HMO even when that HMO tolerates the heat step.
Acidic Drinks
An acidic pH does not automatically rule out an HMO. Test the actual pH, heating time, and shelf life because short pasteurization and long heated storage are very different.
Bars and Baked Products
Record when you add the HMO, the dough or syrup moisture, the product's core temperature, baking time, and final water activity. Oven temperature alone is not enough.
Multi-HMO Blends
Measure each named HMO separately. A total-oligosaccharide result can hide a loss in one HMO or an increase in one of its related carbohydrates.
Choose the HMO Addition Point from Trial Data
Adding the HMO later can reduce heat exposure, but late addition is not always the best manufacturing choice. You must still mix it evenly, control microorganisms, dose it accurately, and make sure the later process steps do not affect it. Adding the HMO before heat may simplify mixing and microbial control, but it exposes the ingredient to more heat.
When both options are practical, compare them in a pilot trial. Measure the HMO after the full process and test whether the HMO is evenly distributed throughout the powder blend or liquid batch. Use the trial results to choose the addition point.
From Sample to Commercial Formula
How to Run an HMO Stability Trial
- Write down the HMO type, exact product specification, target amount, finished-product category, process conditions, package, and planned shelf life.
- Set pass-or-fail limits before the trial. Include the target HMO amount, allowed test variation, related carbohydrates, and relevant physical properties such as moisture, color, or powder flow.
- Test the incoming HMO and keep a sample from that batch. Record its starting assay, moisture, related carbohydrates, and microbiological results.
- Take one sample before processing and another immediately after the step most likely to affect the HMO, such as pasteurization, UHT treatment, spray drying, baking, or fermentation.
- Test the packaged product at time zero to create a baseline for storage. Use the same sampling and extraction method later so the laboratory can compare the results accurately.
- Run real-time shelf-life testing under the planned storage condition. Accelerated testing can reveal problems earlier, but use it to predict normal storage only after you validate the relationship between the two conditions.
- Measure the named HMO with a validated method and check physical and sensory quality at the same time. A stable HMO result does not mean that the product's color, flavor, and texture will also meet your limits throughout shelf life.
Add an Overage Only After You Measure a Repeatable Loss
An overage is an extra amount added to compensate for an expected loss during processing or storage. Set one only after your trials show a repeatable loss and the final HMO amount meets your formulation target and the requirements in your market.
Adding extra HMO before collecting data can hide avoidable processing losses, raise cost, and leave more HMO than intended in the finished product. First find where the loss occurs. Then improve the process or use the trial data to set a controlled overage.
Share Your Processing Conditions
We manufacture six fermentation-derived HMOs. Tell us the HMO type, finished-product format, target amount, pH, heat process, package, storage condition, and planned shelf life. We can discuss a suitable sample and starting test plan with your team.
Discuss an HMO Application