Ref.

Ares(2020)1848056 - 31/03/2020

Natural Enzymes for Ecological Detergents to improve biologically certified Washing

products

Grant Agreement nº: 852970

Deliverable 1.7

Start Date: 1st April 2019

Duration: 24 Months

Deliverable Report
D15: D1.7 Analytical report on ferment and granulate composition

Due date of deliverable: 31/03/2020

Submission date: 31/03/2020
File name: NEEDbioWash_D1.7
Revision number:
Save date:
Document status:
Dissemination level: Pu

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Role Name Organisation Date File suffix
Author Caroline Hennigs NST 31/03/2020 CH
Task Leader Caroline Hennigs NST 31/03/2020 CH
WP leader Caroline Hennigs NST 31/03/2020 CH
Coordinator Caroline Hennigs NST 31/03/2020 CH

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Table of Contents

Summary ............................................................................................................................... 4
1. Introduction ....................................................................................................................... 5
2. Material and methods ........................................................................................................ 6
2.1. Dry Matter Content / Moisture Content ......................................................................... 6
2.2. Ash Content / Mineral Content ..................................................................................... 6
2.3. Protein Content (Kjeldahl) ............................................................................................ 6
2.4. Fat Content (Soxhlet) ................................................................................................... 8
2.5. Determination of carbohydrate content ......................................................................... 9
2.6. Elemental analysis ..................................................................................................... 10
3. Results .............................................................................................................................11

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Summary

The project NEEDbioWash aims to scale-up and demonstrate the production and application of
an organic, GMO-free enzyme product by solid state fermentation which will boost the washing
performance of eco-certified laundry products in a way that they conquer significant market
segments in the laundry detergent market.

NST is about to establish the industrial scale production of 10 tons granulated enzymes per
year. RG will evaluate the production process and the resulting product for the development of
new ecological detergent products.

As the relevant fermentation-, pre-treatment- and granulation parameters are now optimised
according to RG's needs, NST has implement the industrial scale production of the
NEEDbioWash product and application products thereof.
The analysis of the ferment and the granulates composition was carried out by Tecnalia and are
laid down this deliverable.

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1. Introduction

The project NEEDbioWash aims to scale-up and demonstrate the production and application of
an organic, GMO-free enzyme product by solid state fermentation which will boost the washing
performance of eco-certified laundry products in a way that they conquer significant market
segments in the laundry detergent market.

The NEEDbioWash granulate will be assessed according to available guidelines of the

Enzymes REACH pre-Consortium. In order to get the REACH registration a detailed
composition analysis of the product is needed.

NST sent a sample of dry ferment & granulate product to TECNALIA (as in the figure below) to
perform theses analysis.

Fig. 1: Dry ferment & granulated sample (sent by NST)

Two different composition analysis have been carried out:

 Nutritional analysis: according to Regulation (EU) No 1169/2011 of the European Parliament
and of the Council on the provision of food information to consumers the following
parameters have been quantified:
- Water
- Ash
- Proteins
- Total fat
- Total carbohydrate
 Elemental analysis (CHNOPS): quantitative elemental analysis has been performed to
determine present elements:
- Carbon
- Hydrogen
- Nitrogen
- Oxygen
- Phosphorous
- Sulphur

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2. Material and methods

2.1. Dry Matter Content / Moisture Content

In order to calculate nutrient / component content of different products (batches), the dry matter
content is essential to make analytical results for the different parameters comparable. There
are usually different methods based on several standardized methods such as:
 AOAC 930.04 Loss on Drying (Moisture) in Plants

 AOAC 930.15 Loss on Drying (Moisture for Feeds (at 135ºC for 2 hours) – Dry
Matter on Oven Drying for Feeds (at 135ºC for 2 Hours)

The dry matter content of the raw materials is evaluated from the free moisture content. The
latter is measured as weight loss of samples after drying in oven at 105 °C

wt loss on drying [g]

% (w/W) LOD = % (w/w) moisture = 100 x
wt test portion [g]

% Dry matter = 100 - % LOD

2.2. Ash Content / Mineral Content

Ash content refers to the mineral content of a sample and is determined by burning a given
quantity of the sample under prescribed conditions and measuring the residue. It represents the
bulk mineral matter after carbon, oxygen, sulphur and water has been driven off during
combustion. The usual method is based on several standardised methods such as:

 AOAC 930.05 Ash of Plants according to 900.02 or 942.05

 AOAC 942.05 Ash of Animal Feed (at 600ºC for 2 hours)
 AOAC 900.02 Ash of Sugars and Syrups (at 525ºC, several hours)

The dry matter residue is placed in a muffle at 550 ± 10 °C until a white residue (the ashes) is
obtained and weighted.

Weight of test portion [g] – Weight Loss on Ashing [g]

[%] (w/w) Ash = • 100
Weight of test portion [g]

2.3. Protein Content (Kjeldahl)

The determination of the protein content is carried out using the method based on Kjeldahl. The
method is usually implemented using the following standardised methods as reference.

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  AOAC 2001.11 Protein (Crude) in Animal Feed, Forage (Plant Tissue), Grain
and Oilseeds – Block Digestion Method Using Copper Catalyst and Steam
Distillation into Boric Acid
[Applicable to the determination of 0.5–50% Kjeldahl N (3–300% equivalent
crude protein) in forage, animal feed and pet food, grain, and oilseeds, and
applicable to the same matrixes as
976.05(4.2.05),976.06(4.2.06),984.13(4.2.09),988.05(4.2.03),and990.02(4.2.07
); the method does not measure oxidized forms of N or heterocyclic N
compounds.]

Principle
The material is digested in H2SO4 to convert the protein N to (NH4)2SO4 at a boiling point
elevated by the addition of K2SO4 with a Cu catalyst to enhance the reaction rate.

Ammonia is liberated by alkaline steam distillation and quantified titrimetrically with standardized
acid. Aluminum block heaters increase the efficiency of the digestion. The digest must contain
residual H2SO4 to retain the NH3.

Water is added manually or automatically to the digest to avoid mixing concentrated alkali with
concentrated acid and to prevent the digest from solidifying. Concentrated NaOH is added to
neutralize the acid and make the digest basic, and the liberated NH3 is distilled into a boric acid
solution and titrated with a stronger standardized acid, HCl, to a colorimetric endpoint.

The same endpoint detection system e.g., indicator, wavelength) must be used for the
standardization of the HCl and for the analyte. The analyte is referred to as “crude” protein
because the method determines N, a component of all proteins. In addition, N from sources
other than true protein is also determined. (Additional digestion procedures must be used in
order to include N from nitrate.) The amount of protein in most materials is calculated by
multiplying % N by 6.25, because most proteins contain 16% N.

In total the following reactions proceed during the crude protein determination.
K2SO4, Catalyser, Δ
organic substance H2O + CO2 + NH3
2 NH3 + H2SO4 (NH4)2SO4
(NH4)2SO4 + 2NaOH Na2SO4 + 2NH3 + 2H2O
3 NH3 + H3BO3 (NH4)3BO3
(NH4)BO3 + 3 HCl NH4Cl + H3BO3

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 (VS – VB) • M • 14.007
Kjeldahl Nitrogen [%] =
W • 10

Crude Protein [%] = [%] Kjeldahl Nitrogen • F

Where:

VS = volume [ml] of standardized acid to titrate a test

VB = volume [ml] of standardized acid used to titrate reagent blank

M = Molarity of standard HCl

14.007 = Atomic weight of N

W = Weight [g] of test portion or standard

10 = factor to convert mg/g to percent

6.25 = Standard conversion factor for converting Percentages of Nitrogen into Percentages of Protein

2.4. Fat Content (Soxhlet)

Lipid in residues are present in various forms like monoglycerides, diglycerides, triglycerides,
sterols, phospholipids, etc. Lipid matter is soluble in organic solvent and insoluble in water.
Therefore, organic solvents like hexane and petroleum ether have the ability of solubilizing fat
and fat is extracted in combination with the solvent. Later the fat is collected by evaporating the
solvent.

Total fat content is evaluated through Soxhlet extraction with petroleum ether. The method is
usually implemented taking into account the following standardised reference methods:

 AOAC 930.09 Ether Extracts of Plants (according to 920.39B)

 AOAC 920.39 Fat (Crude) or Ether Extract in Animal Feed (wet materials)
 AOAC 954.02 Fat (Crude) or Ether Extract in Pet Food (dried materials)

About some grams of sample are extracted in a Soxhlet apparatus with solvent under reflux for
a certain period. The extraction solvent is collected in a round flask and removed by vacuum
evaporation. The flask with the extracted fat fraction is dry in oven at 150°C to remove moisture,
cooled down in desiccator and weighted.

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Fig. 2: Soxhlet Extraction for the determination of fat content.

The total fat content will be calculated with the following formula.

(m2 – m1) •100

Total fat content [g/100g] =
m0

m0 = weighted sample [g]

m1 = weight of empty extraction flask [g]
m2 = weight of extraction flask with fat after drying [g]

2.5. Determination of carbohydrate content

The amount of available carbohydrates can be calculated if the protein, fat, ash and dry matter
content are known. The following formula is usually used.

Carbohydrate content [g/100g] =100 – m A – m P – m F – (100 – m DM )

mA = Total ash content [g/100g]

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 mP = Total protein content [g/100g]
mF = Total fat content [g/100g]
m DM = Total dry matter content [g/100g]

2.6. Elemental analysis

The elemental analysis of the sample has been carried out according to the following standards:

Table 1: Reference standards for elemental analysis

PARAMETER REFERENCE STANDARD
ASTM Standard Test Methods for Determination of Carbon,
D5373 Hydrogen and Nitrogen in Analysis Samples of Coal and
Carbon Carbon in Analysis Samples of Coal and Coke
EN Solid biofuels – Determination of total content of carbon,
15104 hydrogen and nitrogen – Instrumental methods
Hydrogen DIN ISO Soil quality — Determination of organic and total carbon after
10694 dry combustion (elementary analysis)
DIN EN Characterization of waste - Determination of total organic
Nitrogen 13137 carbon (TOC) in waste, sludges and sediments
DIN EN Solid recovered fuels - Determination of calorific value
15400
Sulphur DIN 51724 Solid mineral fuels - Determination of sulfur content
Oxygen Calculus
DIN EN Water quality — Determination of selected elements by
Phosphorus (P2O5)
ISO11885 inductively coupled plasma optical emission spectrometry
(ICP-OES)

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3. Results

Obtained results are summarized in Table 2.

Table 2: Analytical composition of granulated sample

PARAMETER METHOD UNITS VALUE

Water Gravimetric % 12,00

Ash Gravimetric % 0,30
Total protein Titrimetric % 5,70
Total fat Gravimetric % 1,60
Total carbohydrate Calculus % 80,00
Starch % 60,80
Carbon % 43,20
Hydrogen % 5,40
Nitrogen % 1,20
Sulphur % 0,08
Oxygen % 50,10
Phosphorus (P2O5) % 0,18

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