Reference
Methods Guide
Using proximate analysis
as the basis for NIR calibrations
Introduction
This guide focuses on protein and fat determination, a topic of critical commercial concern to food and pet food ma
nufacturing companies to ensure their products abide by the appropriate laws and legal declaration requirements.
Official reference methods will be discussed, along with their use in creating NIR calibrations that can streamline pro
duction processes and ultimately cut costs in food and pet food industries.
Nutritional analysis began back in 1861, and the methods have continuously been developed, modified, and improved
over the years. The method for the quantitative analysis of macronutrients in food and pet food is called proximate
analysis. To meet industry standards and remain competitive, food-manufacturing companies must utilize reliable
analytical techniques to ensure compliance; this has led to the creation of reference methods. There are reference
methods for determining a whole range of values from samples; however, this guide will specifically describe the refe
rence methods used by different food and pet food industry sectors for protein and fat determination. Once having
understood how to apply reference methods properly, this guide aims to describe how the obtained reference values
become the foundation for building NIR calibrations. NIR analysis can then be used to streamline the entire process of
protein and fat determination and get results in seconds. Other values, such as moisture content, can also be deter
mined rapidly and simultaneously using NIR technology. Refer to each subsection for a more comprehensive list of
analytes covered by NIR spectroscopy.
The primary reference method for protein determination is the tried and tested Kjeldahl method. This method involves
using digestion to release nitrogen from the protein. The nitrogen is converted into ammonia, which is then titrated to
determine the total nitrogen content. The protein content is determined by using a sample-specific empirical protein
factor (conversion factor). Multiplying the total nitrogen content by this conversion factor accurately depicts protein
content. The Kjeldahl method is time-consuming, involving many steps; however, it is a reliable and accepted refe
rence method for accurate protein determination.
Several reference methods are used for fat determination for various samples. The most long-standing and official
method used is the Weibull-Stoldt method, which involves acidic hydrolysis followed by Soxhlet extraction. The food
industry has established several accepted standards based on this method, though other methods exist that can offer
some advantages. Economic Continuous Extraction (ECE) is faster than Soxhlet, though fewer officially accepted
standards exist. Hot Extraction is the fastest and cheapest of the three methods mentioned, and it requires less
solvent. Like with ECE, though, there are fewer official methods. The requirement of performing hydrolysis prior to
extraction when using methods other than Soxhlet is of particular importance. The hydrolysis step is essential as it
releases fat from the matrix, making it available for extraction; this greatly impacts the results and accuracy of the
analysis, more so than the solvent used or extraction method. For these reasons, specific standards governing the
food and pet food industry define the method to be followed for specific sample types. The pet food industry is often
more flexible and likely to use methods other than Weibull Stoldt, as the hydrolysis step is only sometimes required.
There are other methods than those mentioned in this guide, such as alkaline hydrolysis followed by liquid-liquid
extraction (Schmid-Bondzynski-Ratzlaff) for cheese.
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Plant-Based Meat Meat Dairy Bakery
Plant-based foods, such as meat alternatives, are be Fats and proteins are the two main macronutrients in Fat and protein determination are of particular importance This guide will look at protein determination in gluten and
coming increasingly popular due to growing health and meat, and their nutritional value is of high importance to to producers of dairy products. Fat and protein content starch samples and fat determination in cookie samples.
environmental concerns. There are many plant-based al consumer health. The measurement of fat and protein significantly influence the final product in several ways. Proteins and fats are essential to the quality and texture
ternatives to other animal products, such as cheese, milk, content, including connective tissue critical, and helps The industry must carefully balance these factors to en of baked goods. Proteins, especially gluten – as the name
yogurt, butter, and more. The meat alternative market is the food and pet food industry with product development. sure their produce remains fresh and functional over time. suggests – act as the glue that gives baked goods their
experiencing rapid growth, and the role of proteins and An accurate analysis of fat and protein also ensures com structure. Fats provide flavor and prolong the shelf life of
fats in food production is crucial. pliance with regulations. Find out more baked goods.
Find out more Find out more Find out more
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Plant-Based Meat Plant-Based Meat
Protein determination: Vegan Steak Fat determination: Vegan Steak
The role of proteins in meat alternatives is of critical importance as they are used to replicate the taste and texture of Fat plays an important role in plant-based meats as they are used for flavor and to replicate the juiciness of actual
real meat. The nutritional value is also of particular interest as the proteins come from plant-based sources instead of meat. Compliant fat determination is equally necessary for labeling and quality control purposes.
traditional sources of animal origin.
Procedure:
Procedure: The method used for fat determination is the Weibull-Stoldt method. Total fat determination requires a mandatory hy
The Kjeldahl method is an easy and reliable method to determine protein in plant-based meats. Each sample is ho- drolysis step to release the matrix's chemically bound and naturally encased fat. The samples are first hydrolyzed with
mogenized by grinding, depending on the matrix. The sample is then digested using the KjelDigester K-449. Steam the HydrolEx H-506. The fat must then be extracted with a suitable solvent, according to Soxhlet. Extraction was
distillation and boric acid titration are performed with the KjelMaster K-375 and KjelSampler K-377. performed with the FatExtractor E-500 Soxhlet. Afterward, the extract is dried to a constant weight, and the total fat
content is determined gravimetrically.
Parameters Settings
Method parameters
Digestion time 120 min
Hydrolysis 30 min with hydrochloric acid
Digestion temperature 420 °C
Extraction
Distillation time 240 s
Solvent Petroleum ether
Titration type Boric acid
Extraction step 20 cycles (heating level 6)
Sensor type Potentiometric
Rinse step 5 min (heating level 6)
Drying step SmartDrying
Results:
Solvent volume 100 mL
Highlights from the results of this experiment are shown below. For a range of results for several other plant-based
alternatives, click the link and download a copy of the full Application Note that describes the experiment in detail.
Sample Labeled protein content [g / 100 g] Ø determined protein content [%] RSD [%] Results:
Highlights from the results of this experiment are shown below. For a range of results for several other plant-based
Vegan steak 20 19.68 0.95
alternatives, click the link and download a copy of the full Application Note that describes the experiment in detail.
Sample Labeled fat content [g / 100 g] Ø determined fat content [g / 100 g] RSD [%]
Vegan steak 10.8 11.8 0.77
NIR spectroscopy covers analytes such a moisture, fat, protein, carbohydrates, pH, fibre, ash, and color.
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Meat Meat
Protein determination: Sausage Fat determination: Sausage
Protein in meat is of particular interest to athletes and bodybuilders due to the role protein plays in muscle develop For labeling requirements, total fat content must be determined by laboratory analysis and accurately declared.
ment and repair. For people with diabetes, protein helps regulate their blood sugar easing their daily lives. Like with protein, fat content is also concerning for consumers that like to stay fit and healthy.
Procedure: Procedure:
For the analysis of meat, the samples are digested with the SpeedDigester K-439 using a combination of Kjeldahl Economic Continuous Extraction (ECE), also called Twisselmann, is a variation of the traditional Soxhlet extraction
Tablets Titanium and H2O2. The H2O2-assisted digestion protocol can reduce the digestion time by half. Steam distilla method that aims to increase efficiency and reduce the cost of fat extraction. First, the sample is hydrolyzed with the
tion followed by boric acid titration is then performed using the MultiKjel together with the Metrohm Eco Titrator. HydrolEx H-506. Then the ECE is performed on the FatExtractor E-500; this process involves the sample being kept
in the hot solvent vapor while being efficiently rinsed with freshly distilled solvent. The total fat content is determined
Parameters Settings gravimetrically once the extract has been dried to a constant weight.
Digestion time 135 min
Method parameters
Digestion temperature 420 °C Hydrolysis 30 min with hydrochloric acid
Distillation time 180 s Extraction
Titration type Boric acid Solvent Petroleum ether
Sensor type Potentiometric Extraction step 60 min (heating level 5)
Drying SmartDrying
Solvent volume 70 mL
Parameter After standard Kjeldahl digestion
Distillation Time 180 s
Titration Type Boric Acid Titration Results:
Highlights from the results of the experiment are shown below. Click the link and download a copy of the full
Sensor type Potentiometric (pH)
Application Note that describes the experiment in detail.
Labeled fat content Ø determined fat content
Sample RSD [%]
Results: [g / 100 g] [g / 100 g]
Highlights from the results of the standard experiment are shown below. For the results of the accelerated recovery
Cooked sausage, LVU No. 16 – 01j 27.46 27.86 0.25
using H2O2 as a catalyst, click the link and download a copy of the full Application Note that describes the experiment
in detail.
Sample Labeled protein content [g / 100g] Ø determined protein content [%] RSD [%]
Cooked ham 13.677 13.845 0.56
NIR spectroscopy covers analytes such a moisture, fat, protein, CTP, BEFFE, salt, and ash.
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Dairy Dairy
Protein determination: Powdered milk Fat determination: Powdered milk
The protein content of dairy products affects their stability in many ways. When proteins denature, they exhibit several Like protein, fat also significantly impacts dairy production. Fat affects the taste and texture of dairy products.
characteristics, such as a loss of solubility leading to coagulation. Protein content can cause changes in the texture Dairy products, however, are prone to rancidification, which is the autoxidation or hydrolysis of fats and oils when ex
and appearance of dairy products, impacting the quality and storage stability. posed to light, air, moisture, or bacterial action. When the fat in dairy products is broken down, free fatty acids are
produced and, in time, cause the milk to turn rancid. To prevent this process and increase shelf life, spray drying is
used to remove moisture. The high heat used creates an environment that is hostile to bacteria and enzymes, ultimate
Procedure: ly leading to their inactivation.
For the analysis of milk powder, samples are digested with a combination of Kjeldahl Tablets Titanium and H2O2 using
the SpeedDigester K-439. A steam distillation protocol followed by boric acid titration is then performed with the
MultiKjel coupled with the Metrohm Eco Titrator; this results in an easy and fast analysis, and the H2O2-assisted diges Procedure:
tion can reduce the digestion time by half. The fat determination of milk powder involves hydrolysis with hydrochloric acid in the HydrolEx H-506 and a Hot Ex
traction using the FatExtractor E-500. Hydrolysis is a mandatory step for milk powders to obtain accurate results. Hot
Parameters Settings Extraction is the fastest and cheapest method mentioned so far. It requires less solvent than the other extraction
methods. The process is completed by gravimetric determination of fat content after the extract has been dried to a
Distillation Time 180 s constant weight.
Digestion temperature 490 °C
Method parameters
Titration Type Boric Acid Titration
Hydrolysis 30 min with hydrochloric acid
Sensor type Potentiometric (pH) Extraction
Solvent Petroleum ether
Results: Extraction step 5 min (heating level 4)
The results of the experiment are shown below. Click the link and download a copy of the full Application Note that
Rinse step 30 min (heating level 5)
describes the experiment in detail.
Drying step 3 min (heating level 3)
Labeled protein content Ø Determined protein content
Sample RSD [%] Solvent volume 50 mL
[g / 100 g] [g / 100 g]
Milk powder 20.54 20.57 0.20
Results:
Highlights of the results of the experiment are shown below. For further results for several dairy products, click the link
and download a copy of the full Application Note that describes the experiment in detail.
Labeled fat content Ø determined fat content
Sample RSD [%]
[g / 100 g] [g / 100 g]
Milk powder, LVU No. 17 – 4b 24.27 24.35 0.26
NIR spectroscopy covers analytes such a moisture, fat, protein, lactose, pH, salt, and sugar.
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Bakery Bakery
Protein determination: Gluten and starch Fat determination: Cookies
Gluten is a structural protein found in certain cereal grains and is used as a binding and stabilizing agent to give baked The role of fat in baked goods is of particular importance to texture and shelf life, not to mention its influence on taste.
goods a particular structure and texture. Starch is used as a thickener by the food and pet food industry; it is also an Although a number of methods are used for fat determination, none are as thorough as the Weibull-Stoldt method,
essential source of energy. which combines hydrolysis and Soxhlet extraction.
Procedure: Procedure:
The samples are analyzed for their protein content according to the Kjeldahl method. The Kjeldahl method is used to The method used for fat determination, the Weibull-Stoldt method, involves the hydrolyzation of the sample with the
determine organic nitrogen by digesting the sample using the KjelDigester K-449. This is followed by the distillation HydrolEx H-506. The subsequent Soxhlet extraction is performed with the FatExtractor E-500. The extract is then
and the boric acid titration that is performed with the KjelMaster System K-375 / K-376. dried to a constant weight before the total fat is determined gravimetrically.
Parameters Settings Method parameters
Digestion time 125 min Hydrolysis 30 min with hydrochloric acid
Digestion temperature 420 °C Extraction
Distillation time 180 s Solvent Petroleum ether
Titration type Boric acid Extraction step 20 cycles (heating level 5)
Sensor type Potentiometric Rinse step 5 min (heating level 5)
Drying SmartDrying
Solvent volume 100 mL
Results:
Highlights from the results of the Kjeldahl analysis for gluten and starch are shown below. Click the link and download
a copy of the full Application Note that describes the experiment in detail. Results:
Highlights from the results of the experiment are shown below. Click the link and download a copy of the full
Ø Determined nitrogen Ø Determined protein Application Note that describes the experiment in detail.
Sample RSD [%]
content [g / 100 g] content [g / 100 g]
Labeled fat content Ø determined fat content
Gluten 9.498 59.336 0.338 Sample RSD [%]
[g / 100 g] [g / 100 g]
Starch 0.046 0.289 0.619
Cookie, LVU No. 17 – 11 27.47 27.57 0.16
NIR spectroscopy covers analytes such a moisture, fat, protein, gluten, starch, sugar, color, salt,
and baking absorption.
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Conclusion
All the reference methods described give accurate, reliable, and reproducible results; they comply with the official
methods required by law for the determination of protein and fat. Nevertheless, their main pitfall is the time taken to go
through the steps of each procedure. It is not practical for many food and pet food processing plants to continually
perform such time-consuming procedures for quantitative and qualitative identification of sample composition. Thank
fully the accurate results produced by these reference methods also form the basis of reliable NIR calibrations.
NIR spectroscopy is an effective technology that provides fast and cost-effective analysis of fat, protein, and moisture
content, to name only a few parameters. NIR instruments can be installed at-line or on-line at critical stages of the
process to give immediate analytical results or in the lab for quality control. NIR spectroscopy can considerably
streamline a workflow and cut costs significantly. To speed up the implementation of NIR even more, pre-calibrated
applications that already contain a large pool of samples with reference values can be used out of the box. The
combined expertise of NIR and reference methods is important when it comes to calibration development. To learn
more about NIR and take advantage of the benefits of the technology, check out our comprehensive courses and
training sessions.
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