lactose intolerance

Lactose Intolerance & How To Determine Lactose Content In Milk And Milk Products

Simply put, lactose intolerance is the inability of an individual to digest lactose (milk sugar). This complication often leads to undesirable side effects.

Lactose intolerance arises due to the deficiency of the lactase enzyme, which is responsible for the catalytic hydrolysis of the lactose into glucose that can be easily absorbed into the blood.

The problem of lacatse enzyme deficiency is referred to as hypolactasia.

What is hypolactasia? It is when the body cannot produce enough lactase enzyme to digest lactose. This causes lactose intolerance. #lactoseintolerance #foodallergy Click To Tweet

To some extent, hypolactasia may be congenital. In such cases, the victims may not even be able to digest human milk. You should note that lactose intolerance is not milk allergy because it is not an immune response due to the presence of milk proteins.

The manifestations of lactose intolerance may include flatulence, bloating, cramps, vomiting, rumbling stomach/stomach upsets, nausea, and diarrhea.

Normally, most mammals cease lactase production after weaning because they do not need it then. However, humans continue to consume milk long after weaning even into their adulthood.

Some studies have shown that some human populations have developed lactase persistence into their adulthood, enabling them to digest the lactose in milk from cattle and goats.

People who suffer from #lactoseintolerance do not produce lactase enzyme to digest lactose in milk Click To Tweet

These symptoms are as a result of gas production in the small intestine. The lactose sugar cannot be absorbed directly through the walls of the small intestine because it is a disaccharide.

The sugar passes to the colon where the colon bacteria ferments it.

The fermentation process produces large amounts of hydrogen, carbon IV oxide, and methane. The mixture of gases is responsible for all the symptoms manifested by a lactose intolerant individual.

The prevalence of lactose intolerance varies greatly with exposure to dairy products. Those who access dairy products much readily tend to be more tolerant to lactose.

This is because the exposure encourages their bodies to continue producing the enzyme.

On the other hand, those who are less exposed to the dairy products tend to be more intolerant to the lactose because their bodies cease to produce the lactase enzyme.

However, there are exceptions to this observation and may be associated to genetics.

Causes of Lactose Intolerance

Lactose intolerance arises due to deficiency of lactase enzyme. The deficiency of the enzyme can be traced to the following causes

  • Genetic variations: – lactose persistence has been traced to a specific allele in the genome. When this allele lacks in the genome, the body produces minimum or non-existent amounts of lactase. Most of the cases of lactose intolerance in adults have been traced to this primary cause.
  • Acquired deficiency: – considered to be the secondary cause of lactose intolerance. It arises from the injury to the ileum at infancy. The injury may be due to gastroenteritis, intestinal parasites, the effects of chemotherapy, or diarrhea.
  • Congenital deficiency: – this is a very rare genetic disorder, which impedes lactase production right from birth. These individuals cannot digest breast milk. The Finnish commonly exhibit this class of lactase deficiency.

Before the advancement of technology to produce lactose free dairy products and soybean derived baby formulas, the babies born with congenital lactose deficiency never survived.

  • Developmental deficiency: – affects babies born prematurely. It is only temporary and always disappears soon after the baby fully develops.
Causes of #lactoseintolerance include genetics, acquired deficiency, congenital deficiency, & developmental deficiency Click To Tweet

How do you diagnose lactose intolerance?

Listed below are the tests you can carry out to confirm lactose intolerance.

a) Hydrogen in breath test

By far, this is the most accurate test for lactose intolerance. It involves consumption of about 20 grams of lactose made into a solution after fasting for more than 12 hours, preferably overnight.

Since the colon bacteria will metabolize the substrate to produce the gas mixture discussed previously, the gas will be traced in the person’s breath by doing a chromatography test. The procedure takes between two to three hours to finish.

Hydrogen breath test is the most accurate test for #lactoseintolerance Click To Tweet

b) Stool acidity test

This test is best suited for diagnosing lactose intolerance in infants. The infant is given lactose to drink and the stool tested.

Those who are tolerant will digest the lactose and absorb it. Those who are intolerant to lactose will have the lactose reach the colon.

Here, the colon bacteria will metabolize the lactose to produce the mixture of gases. This mixture of gases produces acidity in the stool.

The confirmation of acidity in the infant stool after consuming lactose is a confirmation of intolerance.

c) Stool sugar chromatography

This test identifies traces of sugar in the stool. It is not a very reliable means of identifying lactose intolerance in individuals due to the chemical changes that take place to the lactose in the intestines.

d) Intestinal biopsy

Intestinal biopsy can confirm lactose intolerance in an individual who has elevated levels of hydrogen in their breath after consuming lactose.

By using the gastrointestinal endoscopy equipment, the medical practitioner can be able to identify the deficiency of lactase enzyme in the duodenum.

e) Blood sugar test

The blood sugar levels of the lactose tolerant individuals will show a significant boost after consuming lactose. This is a different observation from the lactose intolerant individuals who will remain level even after consuming lactose.

This is because the tolerant individuals are able to break down the complex sugar (lactose) into simple glucose that the body can absorb.

The intolerant individuals are not able to break down the complex sugar into glucose. The lactose broken down into glucose is responsible for the spike in blood sugar.

After fasting overnight, blood is drawn and tested for blood sugar then the individual is given lactose to ingest. The blood test is repeated after intervals of half an hour, then one-hour, two hours, and three hours.

If the individual is unable to absorb the lactose, there will be insignificant rise in blood sugar.

f) Genetic assay

Lactase persistence in adults is controlled by polymorphic allele in the genes. The procedure involves extraction of DNA material from the blood or saliva of the individual.

Following a matching band of colors, it will be possible to detect whether the person is lactose intolerant or not.

Dairy Products that could trigger lactose intolerance

Lactose is a water-soluble milk sugar. It is often found in the whey portion of the milk when making cheese. The following dairy products contain lactose

Fluid milk: – has high lactose content of up to 9% in human milk, 4.7% cow milk, 4.8% buffalo milk, and 4.9% yak milk.

Cheese: – fermentation and other cheese making processes reduce lactose content in cheeses. Hard cheeses like Cheddar may contain up to 10%  lactose of the whole milk used to make that given cheese sample. Additional cheese ripening processes degrade the lactose to practically nothing after some period. Lactose content is not usually indicated on the cheese labels.

Sour cream: – when made using the traditional methods, the lactose content may be tolerable but modern manufacturers add milk solids into the product raising lactose content.

Yoghurt: – has reduced lactose content due to the action of yoghurt bacteria. These bacteria produce lactase, which catalyzes the hydrolysis of lactose. Traditionally made yoghurt is good for the lactose intolerant because it contains the lactase enzyme produced by the culture bacteria.

Butter and ghee: – has minimal lactose because butter making process excludes most water components of the milk. Since lactose is insoluble in the milk fat, it is separated from the butter during butter processing. Cultured butter will introduce bacteria to break down the remaining lactose in butter. Clarified butter contains minimal amount of lactose, making it good for the lactose intolerant individuals.

Non-dairy products that contain lactose

It is always important to read the labels of the commonly used non-dairy food products as manufacturers often add dairy products to them. Some of the non-dairy products you may find to contain lactose include the following:

  • Bread and other processed wheat products such as waffles, pancakes, biscuits, cookies, doughnuts, frozen waffles and pancakes, toaster pastries, and sweet rolls
  • Processed breakfast cereals, potato chips, corn chips, and other processed snacks
  • Processed meats such as bacon, sausage, hot dogs, and lunch meats
  • Margarine, salad dressings, protein powders and bars
  • Candies, nondairy liquid and powdered coffee creamers, and nondairy whipped toppings

Dealing with lactose intolerance: how to manage the condition

The best medicine for lactose intolerance is to avoid lactose at all costs. If the route of avoidance is out of question, supplementation may be necessary.

It is possible to industrially produce lactase by using Aspergillus spp. fungi. You can obtain the β-galactosidase enzyme as a tablet supplement over the counter without prescription.

One needs to take the supplement at the same time they are taking the lactose-laden food for the supplement to be effective. It works best in an acidic environment; however, too much acid can denature it and render it useless.

Video on natural ways to deal with lactose intolerance (source: Natural Cures)

How to Determine Lactose Content in Milk

We have looked at lactose intolerance, so we know what it is and its causes. We have also seen the various ways victims of lactose intolerance can try to reduce the impact of the problem.

Let us now look at how to determine the lactose content of dairy products.

The required apparatus and reagents:

  • Lactose solution in a burette (2% w/v)
  • Milk sample to be tested
  • Fehling’s solution A (CuSO4.5H2O)
  • Fehling’s solution B (sodium potassium tartrate solution).
  • Measuring cylinder
  • Conical flask(s)
  • Source of heat

Preparing the milk sample for lactose content determination

  1. Into a 100 ml measuring cylinder, add 10 ml of the milk sample and then top it up to the 100 ml mark using cold distilled water
  2. 25 ml of this diluted sample is to be added into the conical flask containing the reagents for the analysis process.
  3. A control sample is prepared by adding 25 ml of distilled water into the conical flask containing the reagents

The Procedure

  1. Add 10 ml of Fehling’s solution A (CuSO4.5H2O) into a conical flask followed by Fehling’s solution B (sodium potassium tartrate solution).
  2. Add 25 ml of the diluted solution (sample) into the conical flask containing solutions A and B.
  3. Heat the contents of the conical flask to boil for two minutes then add three drops of methylene blue indicator into the boiling solution
  4. Add lactose solution into the boiling solution drop wise at intervals of 10 seconds until the blue colour of the methylene blue indicator disappears
  5. Repeat the process for every sample (including the blank)
  6. Record the volume of lactose solution used at the end point of the reaction in each replicate

How to calculate the lactose content

The lactose content is expressed as a percentage of the whole volume. The value varies from one milk sample to the next depending on various factors

Once you have run all the tests and recorded the volume of lactose solution used in every sample, you can determine the value of lactose content in each sample as follows:

Volume of lactose solution used in the control sample = x ml

Volume of lactose solution used in the milk sample = y ml

Percentage lactose (milk sample) = {(x-y)*0.05}/2.5

Where: 0.05 is the % conversion factor for the lactose content and 2.5 is the dilution factor (0.1) multiplied by the diluted sample used in the test (25ml).

You can download this article for more information.

AOAC Official Method 930.28 (2000) for Determination of Lactose Content in Milk (Munson-Walker Gravimetric Method).

  • Dilute 25 g of the test portion with 400 mL water in 500 mL volumetric flask.
  • Add 10 mL CuSO4 solution, and 7.5 mL KOH solution of such concentration that 1 volume is just enough to completely precipitate the Cu as hydroxide from 1 volume of the CuSO4 solution. (Instead, 8.8 mL 0.5M NaOH may be used. After addition of alkali solution, mixture must still be acid and contain Cu in solution.)
  • Dilute to volume, mix, filter through dry filter, and determine lactose in aliquot of filtrate.
  • From the aliquot obtain weight lactose equivalent to weight Cu2O.

LACTOSENS® Assay: A Rapid Computerized Lactose Content Determination Method

This section was written by a guest

This section of the article was contributed to by Josef Michael Kienböck of Direct Sens. Direct Sens is a university-connected venture with a strong scientific basis on life science technologies. You can see their projects here: http://www.directsens.com/lactosens/

Milk is a very rich source of lactose, which causes lactose intolerance to over 70% of adult population globally. Lactose intolerance affects people of all ethnicities/races. Lactose content determination for product labeling purposes is critical owing to the serious nature of lactose intolerance.

lactose intolerance statistics

For instance, Virtual Medical Center indicates the following:

Primary lactose intolerance affects between 7 to 20% Caucasians, 65 to 75% African descent, 90% in certain Asian populations and about 70% Australian/Aboriginal populations.

Putting these figures into perspective can be very depressing, especially when considering dairy products as a means to fight world hunger and malnutrition.

Increasingly, many people are pushing for dairy products with reduced lactose content. Through technology, several advances have been made.

For instance, fermented products such as yoghurt and cheeses have proven very popular. This is because they do not elicit similar reactions as fresh milk in people with lactose intolerance.

A similar story cannot be said about fluid milk. Fluid milk forms the bulk of dairy products consumed globally by volume.

But there is a problem,

Fluid milk naturally has high content of lactose making it unsuitable for many who are lactose intolerant.

Determination of lactose content is usually a tedious process involving a series of meticulous steps. The process is often expensive and time consuming. Most labs do not have the sophisticated equipment required for these tests (HPLC or GC) hence many processors often opt out.

There needs to be a cheap, accurate and reproducible method of determining and controlling the lactose content in fluid milk and other dairy products.

Luckily, there is a new rapid and relatively cheap lactose content determination method that can be adopted for both industrial and artisanal use.

LACTOSENS® Assay: A computerized rapid, accurate and cheaper lactose content determination method for dairy products.

Imagine, if you could measure the precise residual lactose level in your dairy products in real-time – at every stage of production and with a sensitivity of 0.01%.

LACTOSENS®, based on amperometric biosensor technology, is the ideal solution for process monitoring and quality control. It provides a quick and efficient route to market and shortening lead times in the booming lactose-free segment.

With the globalization of dairy markets, “lactose-free” has become a solution to meet the needs of over 70% of the world’s population!

Infants are born with the ability to produce lactase, but with increasing age the production of lactase reduces or ceases.

In order to avoid inconveniences resulting from lactose non-digestion, many consumers are turning to “lactose-free” dairy products. This allows them to commonly use milk as an important source of protein, minerals and vitamins.

With this increasing importance of “lactose-free” products, the relevant threshold for “lactose-free” labeling has been revised from the former 0.1g/ml to the more stringent standard of 0.01g/ml.

This readjustment to the lower threshold is aimed at people with severe lactose intolerance. The adjustment presents even a bigger challenge for dairies. Existing methods for precise control and verification of a 0.01% lactose threshold are quite laborious, slow and expensive, especially when not executed in-house.

Thus, dairies may have to wait for days to get a result for product release – or release batches based on “typical” results. This means such dairies will not have traceable batch-specific concentration data available for all productions.

Accuracy: How LACTOSENS® compares with HPLC.

The LACTOSENS® 0.01% assay – recently validated against HPLC – here opens a unique access for dairies and even processing dairy farmers to a fast and precise method for lactose concentration measurement.

The test involves a direct assay on a biochip that very specifically measures lactose in mere minutes in milk and dairy products as yogurt, sour cream, fresh cheese and mozzarella.

The graph below shows how the accuracy of LactoSens compares to the HPLC for lactose content determination.

lactosens vs HPLC accuracy

The Working Principle of LACTOSENS® Assay

What’s the secret functional principle of this fast and precise LACTOSENS® biosensor?

Here is how it works.

  • 100μl of the diluted sample is applied to the disposable test strip so that the circular sensor area carrying the immobilized enzyme is completely covered with sample.
  • The electrochemical measurement is based on a principle known in science as amperometry:
  • During the measurement, lactose in the sample is oxidized by the highly specific, genetically optimized enzyme to lactobionic acid (a sugar acid – disaccharide – of gluconic acid and galactose). The electrons resulting from the oxidation are measured directly by the potential-active LACTOSENS® reader.
  • Finally, the software transmits the analytical signal according to the calibration function into a lactose concentration.

You can check out the LactoSens product documentation for more details.

How to operate the LACTOSENS® machine for lactose content determination in dairy products

LACTOSENS® TEST – SO SIMPLE & FAST 1. Dilute the milk sample with the provided ready-to-use buffer 2. Scan the QR code on the sensor bag 3. Unpack the sensor and insert it into the reader 4. Pipette 100μl sample onto the sensor area 5. Press „START“                                                         produces results in < 1 minute

The video below illustrates how LACTOSENS® machine is used to determine the lactose content of dairy products

Certification by NordVal International

The LACTOSENS® trace analysis detection is so precise, that the NordVal International Institute of the Technical University of Denmark has certified the LACTOSENS® biosensor – in the sensitive range between 0.1% and 0.008% – as equivalent to the lactose reference method HPLC:

The high-performance liquid chromatography requires expensive equipment and qualified personnel.

Control studies were done at two ISO 17025 accredited laboratories. The results validated the LACTOSENS® biosensor against the HPLC method in ruggedness, specificity of detection, stability testing and batch-to-batch variation (traceability), limit of quantification, precision and internal reproducibility (reliability).

This NordVal validation study confirms the LACTOSENS® biosensor as unique rapid lactose test. It detects lactose at levels of 0.008% and produces comparable results to the HPLC method.

You can find the NordVal Certificate for LACTOSENS® here.

10 Justifications for dairies to adopt LACTOSENS® assay:

  1. IMMEDIATE: Release your products promptly as soon as in-house testing confirms the lactose concentration is at the claimed level.
  2. RAPID: The measurement process completed in less than one minute and fast product release save both labour time and money.
  3. TRACEABLE: Document low lactose or lactose-free claims for every batch produced. Protect your brand by enhancing traceability. This guarantee will solidify the trust your customers have in your brand hence better quality relationships.
  4. EASY TO USE: There are very low training requirements – no special skills are needed. A permanent on-screen guide leads the analyst through the process.
  5. SPECIFIC: There is no interference from other sugars, such as glucose or galactose. This is because high-selectivity biosensor is used in the equipment.
  6. ACCURATE: LACTOSENS® has a high sensitivity and detects residual lactose levels lower than 0.01%. Furthermore, lactose is directly measured instead of its decomposition products.
  7. RELIABLE: LACTOSENS® is the only lactose rapid test validated against the HPLC reference method. The comparison to HPLC in terms of accuracy and reproducibility up to 0.008% lactose has been certified. Therefore, LACTOSENS® can easily be integrated into any quality management system.
  8. FLEXIBLE: No initial preparation and calibration of the biosensor is necessary: As a portable handy device, LACTOSENS® is always ready for use and connectable with any notebook.
  9. BROAD APPLICATION RANGE: LACTOSENS® works with a broad range of lactose-free and low lactose dairy samples like milk, cream, yoghurt, fresh cheese, mozzarella.
  10. DAIRY EXPERT SUPPORT: The Chr. Hansen dairy team is always on hand to provide advice – wherever in the world you may be.

What the experts are saying about LACTOSENS®:

‘Lactosens is ideal for both process control and product release in the dairy industries,’ explains Jes Jensen, Director of Testing and Equipment at Chr. Hansen. ‘It is the fastest and easiest method available, revolutionizing the way in which the dairy industry analyzes lactose.’

How to Get the LACTOSENS® Machine

All customer orders from different parts of the world are fulfilled through Chr. Hansen exclusively. Chr, Hansen is a leading well-known dairy specialist with sufficient sales force and offices covering all global markets.

You can get in touch with them to inquire about LACTOSENS® https://www.chr-hansen.com/en/contact.

References

Behrendt, M., Keiser, M., Hoch, M., & Naim, H. Y. (2009). Impaired trafficking and subcellular localization of a mutant lactase associated with congenital lactase deficiency. Gastroenterology, 2295-2303.

Beja-Pereira, A., Luikart, G., England, P. R., Bradley, D. G., Jann, O. C., Bertorelle, G., . . . Erhardt, G. (2003). Gene-culture coevolution between cattle milk protein genes and human lactase genes. Nature Genetics, 311-313.

Eddleman, H. P. (1999, February 6). Composition of Human, Cow, and Goats Milk. Retrieved from Goatworld.com: https://www.goatworld.com/articles/goatmilk/colostrum.shtml

Roy, B., Nwakakwa, S., & Khurana. (2006, July 5). Lactose Intolerance. Retrieved from eMedicine.

Swagerty, D. L., Walling, A. D., & Klein, R. M. (2002). Lactose Intolerance. American Family Physician, 1845-1850.

Swallow, D. (2003). Genetics of lactose persistence and lactose intolerance. Annual Review of Genetics, 197-219.

Vesa, T. H., Marteau, P., & Korpela, R. (2000). Lactose intolerance. Journal of the American College of Nutrition, 165-175.

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Responses

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  1. I am having trouble understanding your procedure. I understand the process you describe in steps #1-3 of preparing both the unknown milk sample and the control. For steps 5 and 6 it states add 10ml of Fehling’s A and B solution to a flask and then add 25 ml of lactose solution, add methylene blue and boil. Then tritrate this flask with lactose solution. Why are steps 5-9 needed. Why can’t you titrate the flasks from steps #2 (milk sample) and #3 (control ) and determine the volume of lactose used for each titration.
    How do steps # 5-9 relate to your procedure?

    1. Hi Christopher,
      The first three steps are for preparing the milk sample. The analytical procedure begins at number 5 (I have edited it so that the procedure stands out on its own sub-section).

  2. I am unable to understand how to make the lactose solution, what is the exact procedure? Do we need lactose in some form? Because I don’t have any lactose.

  3. I have lactose and it is not liquid, I’ve read the manufacturers label and there’s is no instructions on how to make it into a solution. How do I make a lactose solution?

    1. Hi Megan, your comment was filtered by Akismet. Explains why it took so long to get a response.
      The dilution rate will depend on the purity of the substance, so, I will not tell you directly to add a given amount to a certain volume of distilled water. However, by convention, you make 2% of the pure lactose solution. Make sure you have pure lactose powder. 2%w/v means that you add 2g of lactose powder into 100ml of distilled water.

  4. the principle behind the estimation is not clear
    what kind of complex will be formed? what will get reduced what will get oxidized? how will the colour change? the basic concept is what?

  5. Can you please explain the calculation formula. Where is this 0.05 and 2.5 coming from? Thank you.

  6. Hi Kevine,
    I recently completed the experiment at school and was having some problems with the formula supplied at the end, is the percentage lactose supposed to be negative (when calculating using averages)?

  7. Apologies, but I wanted some clarity on whether the instructions meant to use 10ml of Fehling’s solution A and 10ml of Fehling’s solution (10ml of each) or if it was meant to be a 10ml total of both (5ml each)

  8. When the Instructions say ”until the blue colour of the methylene blue indicator dissappears” will the solution completely decolourise to give a clear solution

  9. So if i had to use a dilution factor of 0.05 for the milk sample, would the formula still work as long as i replaced the dilution factor 0.1 for 0.05?

  10. Hey, I think your method is very helpful and interesting. I’m just wondering how Fehling’s solution A and Fehling’s solution B help to determine the concentration?

  11. I’m confused on what the control sample contents are. Is it 5 ml of Felhing A and 5 ml of Fehling B with 25 ml of distilled water? And the sample solution should have Fehling A (5ml) and Fehling B (5ml) along with 25ml of diluted milk sample solution? Also, I am making rochelle crystals at home and will crush them and put them in water for the sodium tartrate solution (Fehling B) to be made since it isn’t available at my school. Will that work?

    1. The control does not contain any sample. It is just a blank (with distilled water). As for the crystals you are making at home, I am not sure of their purity. Maybe you can do that as a project then come back and tell us what you got.

  12. This method didn’t give me accurate results. However it worked much better when i used 5ml milk and 95ml water. This gave me much more accurate results. However I am having difficulty with the formula now that I have changed the dilution. How would I use the formula now?

    1. That will need a workaround to adjust. How did your figures compare with the conventional method and how did you know your results are more accurate if you could not calculate the % lactose? Did you compare results with a lactoscan output?

  13. Can u please explain what happens during the reaction like what kind of complex will be formed? what will get reduced? what will get oxidized? how will the colour change?

  14. I didn’t understand what is oxidized and what is reduced and I opened the article you added and I still didn’t understand, can you please further explain the reaction between lactose and Fehling A and B

    1. The milk sugars (lactose) will form a complex with Fehling’s solution, which in the presence of an indicator changes colour (turns colourless) at the end point of the reaction (when all the lactose has been used up). The quantity of reagents used is then used to calculate the quantity of lactose in the milk (molar comparisons).

  15. Hello, In step 3 when content in flask is boiled turns brown in color from blue and on adding methylene blue its color changes to colorless then how can i go for titration can you help me n tell the reason behind it?

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