công nghệ lên men calvados

BACH KHOA UNIVERSITY

FERMENTATION TECHNOLOGY REPORT

CALVADOS
Group: 2
Student
Name

Student number

Huynh Kim Chi

1510287

To Phan Chieu Dan

1510620

Instructor: Le Van Viet Man

HO CHI MINH CITY, 04/2018
CONTENT

DANH MỤC HÌNH


DANH MỤC BẢNG


INTRODUCTION
Calvados is an apple brandy from the Normandy region in France. Apple orchards and
brewers are mentioned as far back as the 8th century by Charlemagne. The first known
Norman distillation was carried out by Gilles Picot, Lord de Gouberville, in 1553, and the
guild for cider distillation was created about 50 years later in 1606. In the 17th century,

the traditional cider farms expanded, but taxation and prohibition of cider brandies were
enforced elsewhere than Brittany, Maine, and Normandy. The area called "Calvados" was
created after the French Revolution, but eau de vie de cidre was already called calvados
in common usage. In the 19th century, output increased with industrial distillation and the
working class fashion for café-calva. When a phylloxera outbreak in the last quarter of
the 19th century devastated the vineyards of France and Europe, calvados experienced a
"golden age". During World War I, cider brandy was requisitioned for use in armaments
due to its alcohol content. The appellation contrôlée regulations officially gave calvados a
protected name in 1942. After the war, many cider houses and distilleries were
reconstructed, mainly in the Pays d'Auge. Many of the traditional farmhouse structures
were replaced by modern agriculture with high output. The Calvados appellation system
was revised in 1984 and 1996. Pommeau got its recognition in 1991; in 1997, an

appellation for Domfront with 30% pears was created.

5


I. Materials
I.1 Main material: Apple

Not any apples can also make Calvados, to distilled the precious Calvados bottles
with beautiful scent and color, we need quality apples of Normandie, an area in west of
French.

Figure 1. 1 Normandie apple
The apple varieties, classified in 4 categories: bitter, acidulous, sweet or bittersweet,
don't mature at the same time and are not intended for the same kind of production.
Table 1. 1 Normandie apple classification
Sweet
Sharp
Bittersweet
Bitter

Bedan, Bisquet, Douce coetligne, Tete de brebis
Binet rouge, Clos renaux, Douce moen
Avrollers, Judaine, Judeline, Juliana, Petit jaune

Frequin rouge, Kermerrien, Peau de chien

The most common are: Antoinette, Frequin Rouge, Bisquet, Moulin à Vent (bitter and
bittersweet apples, 70% of the production), Bedan, Noël des Champs (sweet apples,
20%), Rambaud, René Martin (acid apples, 10%).
The early apples, collected between September 15 and October 30 are generally
processed within 48 hours into juice which will be used for Calvados production.
The late apples, collected until December 15 can mature in openwork wooden boxes
(palox) in a frost-free place during 3 to 4 weeks. Sugars and flavours accumulate and
constitute the main components of a quality cider and quality pommeau production.

6

Table 1.2 Nutrition of Normandie apple

Nutrition Facts
Finest, Tart, Normady Apple and Calvados
Serving Size: 1 tart ( 3,5 oz)
 Amount Per Serving

Calories
Calories from Fat

256 Cal
63 Cal
% Daily Value
11%
_%
_%
%
14%
4%
%
6%

Total Fat
Saturated Fat
Cholesterol
Sodium
Total Carbonhydrate
Dietary Fiber
Sugar(Added)
Protein

7g
0,6g
0 mg

456mg
41g
1g
0g
3g

Vitamins
Vitamin A
Vitamin B6
Vitamin B12
Vitamin C
Vitamin D

Vitamin E
Vitamin K

0 IU
0.1mg
0.2mg
1.3mg
0 mg
0 mg
2.5 mcg

%

%
%
%
%
%
%

Minerals
Calcium (Ca)
Copper (Cu)
Iron(Fe)
Magnesium (Mg)

Manganese (Mn)
Potassium (K)
Phosphorus (P)
Selenium (Se)
Zinic (Zn)

22mg
0.1 mg
0.8 mg
19 mg
0 mg
139mg

84mg
5mg
0.8mg

%
%
%
%
%
%
%
%

%

7


Table 1. 2 Standard of Normandie apple
Properties
Shape

Description
Apple fruits must be free of decay or rot and well-matured. Unripe or
immatured fruits should not be used because they are rich in starch,

acids
and astringent compounds while low in sugar and flavoring
compounds.
Overly matured fruits can be low in fresh and fruity flavor, difficult for
further processing and also difficult to clarify.
Flavor
No strange flavor
Color
Color of apples are uniformly pink
Total soluble 12-13°Brix
solid content
I.2 Supplementary material

I.2.1 Water
 Function:

Water involves in most of the operations in the manufacturing process of calvados.
Water is divided into: water used to wash the material, equipment and water employed as
a component in the final product . Therefore, water quality is important.
 Standards:

Table 1. 3 Standard of water
Target
Physical
Flavor

Clearness (Dienert)
Color (cobalt color scale)
Chemical
pH
Residue levels fixed (600oC)
Permanent hardness
Full hardness
CaO
MgO
Fe2O3
MnO
BO4-3

SO4-2

Requirement
No
100 ml
5o
6.0-7.8
75-150 mg/mL
7o
Lower than 15o
50-100m g/L
50 mg/L

0.3 mg/L
0.2 mg/L
1.2-2.5 mg/L
0.5 mg/L
8


NH4+
NO2NO3Pb
As
Cu
Zn

F
Microbial
Total aerobic microorganisms
Coli index
Coli standard number
Pathogenic microorganisms

0.1-0.3 mg/L
0
0
0.1 mg/L
0.05 mg/L

2 mg/L
5 mg/L
0.3-0.5 mg/L
<100 cfu/mL
<20 cfu/L
>50 mL
0

I.2.2 Sucrose
 Function

Sucrose is used to supplement the fermentation medium, in order to adjust the sugar

content to that required for fermentation. It is usually refined sugar (RS) or crystalized
sugar.
 Chemical properties

Chemical formula: C12H22O11.
Molecular weight: 342.30 g/mol.
Sucrose is a disaccharide, a molecule derived from two simple sugars
(monosaccharides). In sucrose, the monosaccharide building blocks are fructose and
glucose. The splitting of sucrose is a hydrolysis reaction. The hydrolysis can be induced
simply by heating an aqueous solution of sucrose, but more commonly, catalysts are
added to accelerate the conversion.
Inverted or invert sugar in syrup is a mixture of glucose and fructose; it is obtained by

splitting the sucrose into these two components. Compared with its precursor, sucrose,
inverted sugar is sweeter-tasting and its products tend to retain moisture and are less
prone to crystallization.

9


Request of sucrose is colorless solution or pale yellow, with no strange odors affecting
the product's natural scent. The different designations for syrups range from light fruit
juice syrups to extra heavy syrups. Their designations followed by their Brix
measurement are: extra heavy syrup (E): 22 to 35 o, heavy syrup (H): 18 to 22o, light syrup
(L): 14 to 18o, and light fruit juice syrup or water (W) <14 o. Syrup strengths may be

verified using a refractometer or a Brix hydrometer.
 Physical properties

Apperance: white solid.
Density: 1.587 g/cm3.
Solubility in water: 2100 g/L (at 25°C).
 Standard
Table 1. 4 Standard of refined sugar (TCVN 6958-2001)
Number

Standard

Level

1

Pol degree (% dry weight)

≥99.80

2

Reducing sugar content (%
mass)

≤0.3

3

Ash (% mass)

≤0.03

4

Moisture (% mass)

≤0.05

5

Colour (ICUMSA)

≤30

I.2.3 Additional substrate and growth factor
a) Diammonium phosphate
 Function:


•
•
•

Diammonium phosphate (DAP) is an inorganic nitrogen that yeasts can assimilate.
The addition of this supplemental nitrogenous compound will improve the fermentability
of the yeasts.
Chemical properties
Chemical formula: (NH4)2HPO4
Composition: 18% N, 46% P2O5 (20% P)

Molecular weight: 132.07 g/mol
10


Figure 1. 2 Diammonium phosphate

•
•
•
•


Physical properties
Colorless crystal, saline taste.
Density: 1.619 g/cm3.
Water solubility (20°C): 588g/L
Solution pH: 7,5 to 8
Standard
Table 1. 5 Standard of Diammonium phosphate

Quality criteria
Assay
Arsenic

Limit
≥ 96.0%
≤ 3 mg/kg

Fluoride

≤ 10 mg/kg

Free acid

≤ 4 mg/kg

b) Thiamine
11


 Function:

Thiamine (vitamin B1) is a growth factor of yeast which involves in decarboxylation
of pyruvate, oxidation and dearboxylation of oxo acids. Thiamine preparation is added to
the medium to encourage vigorous alcoholic fermentation. Adequate concentrations of
thiamine reduce the synthesis of carbonyl compounds binding to sulfur dioxide, thereby
diminishing the amount of SO2 needed to control spoilage organisms. Thiamine also
reduces the concentration of higher alcohols produced during fermentation.

 Chemical properties
Chemical formula: C12H7ClN4OS
Thiamine is soluble in water and glycerol and is practically insoluble in extremely
low organic solvents. It is stable at acidic pH, but unstable in alkaline solution.

Figure 1. 3 Thiamine

 Physical properties

Thiamine is unstable with heat, but stable when stored frozen. It is unstable when
exposed to ultraviolet light and gamma radiation.
 Standard

Table 1. 6 Standard of Thiamine

Criteria

Value
12


Assay

≥ 99%

Moisture content

≤ 5%

pH

2.7 ‒ 3.4

Ash

≤ 0,1%

Sulfate

≤ 300 ppm

Nitrate

0

Heavy metal

≤ 20 ppm

c) Potassium Metabisulfite
 Function

Potassium metabisulfite is employed as an antimicrobial and antioxidant agent by the
generation of sulfur dioxide in aqueous solution.
Sulfur dioxide has the potential to bleach pigments, suppress oxidized odour and
inhibit a wide range of microbes.
 Chemical properties
Potassium metabisulfite (potassium pyrosulfite) is an inorganic compound with the
chemical formula K2S2O5.
Molecular weight: 222.31 g/mol.

13


Figure 1. 4 Potassium Metabisulfite

•
•
•


Physical properties

Specific gravity: 2.34 g/cm3
Soluble in water (250 g/L at 20oC), insoluble in alcohol.
Potassium metabisulfite is gradually oxidized in air to give sulfate.
Standard
Table 1. 7 Standard of potassium metabisulfite

Quality criteria

Limit

Assay

≥ 96.5%

Moisture content

≤ 3.5%

Total
subtances

≤ 0.05 %

insoluble

Arsen

≤ 1ppm

Lead

≤ 2ppm

Iron

≤ 10ppm

Selenium

≤ 5ppm

d) Sodium Bentonite
14


 Function

Sodium bentonite is employed as a fining agent in wine treatment to eliminate

unstable proteins which are responsible for protein casse. This operation will improve the
quality of the final product.
 Chemical properties

Chemical formula: Al2H2Na2O13Si4
Molecular Weight: 422.286 g/mol.
Montmorillonite is structured in separate flakes, giving it remarkable colloidal
properties. Montmorillonite, which swells considerably in aqueous media, has a large
adsorption surface and negatively charged.

•
•

•
•
•
•
•

Physical properties
Appearance: Granular powder, dust
Colour: Grey, green
Odour: Odourless
Solubility: Insoluble in water
Melting point (°C): >450°C

Relative density: 2.7
pH-Value: 9 - 10
.

15


Figure 1. 5 Sodium bentonite
 Standard

Table 1. 8 Standard of sodium bentonite

Quality criteria

Limit

Coarse particle

Not more than 0.5% of
sample
is
retained on 75μm sieve

pH

8.5 – 10.5

Arsen

≤ 5 mg/kg

Lead

≤ 4 mg/kg

Aerobic plate count

≤ 103 cfu/g

Escherichia coli

Absence/25g

e) Diatomite
 Function

Diatomite is employed as a filter aid in wine making.
 Chemical properties

16


Diatomite (kieselguhr or diatomaceous earth) is a siliceous sedimentary rock,
resulting from the accumulation of microscopic fossil algae shells, or diatomaceous earth,
with dimensions ranging from a few to several hundred micrometers

Figure 1. 6 Diatomite

•
•


Physical properties
Density: 0.45 g/ cm3
pH: 6-7
Standard
Table 1. 9 Standard of diatomite

Criteria Quality

Description

Product form

Powder

Color

White to brown

Density

0.45 g/cm3

pH

6-7

Moisture content

≤ 1%

I.2.4 Enzyme preparations

Apple peel contains cellulose and lignin, which are insoluble in water. The pulp
consists of cell wall, the middle lamella which is a specialized region associated with the
17

cell walls of plants is rich in pectins, water and the nutritional compounds. The seed
contains phenolic compounds which are contribute to the bitter taste of the juice.
If we want to extract completely the apple juice, we need to break down the cell wall
and break down the middle lamella. Thus, after crushing and de-stemming we need to use
cellulase and pectinase for a complete degradation of cellulose and pectin.
a) Cellulase preparation
 Function

Cellulase preparation is employed to break down cellulose, the main component of
cell wall in the pulp particles. This operation is aimed to increase the extraction yield and

prepare for the treatment with pectinase.
Cellulase is a hydrolase produced primarily by fungi and bacteria. This enzyme
catalyzes the hydrolytic reaction of the β-1,4-glycosidic bond in cellulose, hemicellulose,
lichenin and β-D-glucan with the participation of water. Cellulase breaks down the
cellulose molecule into monosaccharide (D-glucose) or shorter oligosaccharides.

•
•
•
•
•
•



Commercial cellulase preparation
Selected cellulase preparation: Celluclast® 1.5 L.
Supplier: Novozymes, Switzerland.
Ingredients: endoglucanase, potassium sorbate, sodium chloride and sorbitol.
Microbial source: filamentous fungus Trichodema reesei.
Optimal pH: 4 ‒ 6.
Optimal temperature: 50 – 65°C.
Standard
Catalytic activity

≥ 700 EGU/mL

Color

Brown

Odor

Slightly fermented

Physical state

Liquid

Specific weight

1.22 g/mL

Total plate count

≤ 100 cfu/g

Coliform

≤ 30 cfu/g

Escherichia coli

Absent/25g

Salmonella

Absent/25g
18

Lead

≤ 5 ppm

Arsen

≤ 3 ppm

Cadium

≤ 0.5 ppm

 Determination of cellulase activity (EGU ‒ Endoglucanse Unit):

A substrate solution containing 34.0 g/L CMC (carboxymethyl cellulose) in 0.1 M
phosphate buffer, pH 6.0. The enzyme sample to be analysed is dissolved in the same
buffer. 10 mL of substrate solution and 0.5 mL of enzyme solution are mixed and
transferred to a vibration viscosimeter. thermostated at 40°C. One endoglucanase unit
(EGU) is defined as the amount of enzyme that reduces the viscosity to one half under
these conditions.
b) Pectinase preparation
 Function

Pectinase is employed to degrade pectin in the middle lamella of the apple flesh

improve the extraction of the juice.
Endo-polygalacturonase (EC 3.2.1.15) catalyzes the hydrolytic reaction of α-1,4glycosidic bond in the middle of the pectic acid chain. This result in the reduction of the
juice viscosity.
Pectinlyase (EC 4.2.2.10) catalyzes the hydrolytic reaction of α-1,4-glycosidic bonds
in the middle of the pectic acid chain without water involvement to release the product
with a double bond.
Pectinesterase (EC 3.1.1.11) catalyzes the hydrolytic reaction of the ester linkage in
the pectin molecule to give pectate and methanol.

•
•
•

•
•
•

Commercial cellulase preparation
Selected cellulase preparation: Pectinex ultra SPL
Supplier: Novozymes, Switzerland.
Ingredients: endo-polygalacturonase, pectin lyase and pectinesterase.
Microbial source: filamentous fungus Aspergillus aculeatus.
Optimal pH: 4.5.
Optimal temperature: 40 – 45°C.

19


Figure 1. 7 Pectinase preparation
 Standard

Table 1. 10 Standard of pectinase preparation

Quality criteria

Limit

Color

Brown

Odor

Slight fermented

Density

1.16 g/mL)

Catalytic activity

≥ 9,500 PGU/mL

Total plate count

≤ 500 cfu/g

Coliform

≤ 30 cfu/g

Escherichia coli

Absent/25g

Salmonella

Absent/25g

 Determination of polygalacturonase activity by viscometric assay:

20

One polygalacturonase unit (PGU) is the amount of enzyme that will cause a change
in relative fluidity of 0.1/sec in a defined sodium polypectate substrate under the
conditions of the assay.
II. Starter culture
II.1 Wine yeast Saccharomyces cerevisiae
II.1.1
Function

The yeast Saccharomyces cerevisiae is responsible for performing alcoholic
fermentation.
II.1.2

Introduction
• Classification

Figure 2. 1 S. cerevisiae
•

Description: S. cerevisiae cells are round to ovoid, 5–10 μm in diameter. It reproduces by
a division process known as budding
21


•

Growth Conditions: The optimum temperature for growth of S. cerevisiae is 30–35 °C
(86–95 °F).
• Nutritional Requirements
Carbon source: All strains S. cerevisae can utilize glucose, fructose sucrose and
maltose but not five carbon sugars.
 Nitrogen source: The yeast S. cerevisae cannot assimilate atmospheric
nitrogen. The common nitrogen sources are inorganic compounds (ammonium
salts, urea) and organic compounds (amino acids, peptides, amines, purine and
pirimidine). Polypeptides and proteins do not participate in S. cerevisiae
growth, since this yeast cannot hydrolyze these substances. Moreover, S.
cerevisae cannot utilize nitrate and nitrite salts.

 Minerals: Two minerals mainly focused are phosphorus and sulfur. The
phosphorous source for S. cerevisae is phosphate anion. The sulfur sources for
this yeast are organic compounds (methionine, cysteine, glutathione) and
inorganic salts (sulfate, sulfite and thiosulfate).
 Growth factors: Vitamins, purine, pyrimidine, polyamine, nucleoside and some
lipids.
Commercial starter cultures


II.1.3

Brand name: Lalvin EC1118

• Original: LALLEMAND Inc. (Canada)
• Description: Active Dry Yeast, a strain of Saccharomyces cerevisae
• Application: Useful for a wide range of applications, including wine and fruit cider
fermentations.
• Properties
 Desirable fermentation temperature: 10-30°C.
•



Alcohol tolerance 18% v/v *subject to fermentation conditions.



Low relative nitrogen demand (under controlled laboratory conditions)



Short lag phase and high fermentation vigour. Cooling may be required to
control this high vigour.



Very low production of H2S



Low production of SO2 binding compounds.



Killer factor active.



Low foam producer.

•

Packaging: 500g
22


Figure 2. 2 Commercial S.cerevisiae
•

Instruction for use
Procedure for 1000L ferment.

1) Add 300g of Go-Ferm Protect® / Go-Ferm Protect Evolution™ to 5L of 40-43°C
clean, chlorine free water. Stir until an homogenous suspension free of lumps is achieved.
2) When the temperature of this suspension is between 35-40°C, sprinkle 250g of
yeast slowly and evenly onto the surface of the water, whilst gently stirring. Ensure any
clumps are dispersed.
3) Allow to stand for 20 minutes before further gently mixing.
4) Mix the rehydrated yeast with a little juice, gradually adjusting the yeast
suspension temperature to within 5-10°C of the juice/must temperature.

•
•

5) Inoculate into the must.
Storage: should be stored dry, best practice between 4-12°C and the vacuum packaging
should remain intact.
Quality

23


Table 2. 1 Quality of commercial S.cereviae

II.2 Lactic acid bacterium Oenococcus oeni
II.2.1

Function

Oenococcus oeni is responsible for performing malolactic fermentation.
II.2.2
Introduction
• Classification

24


Figure 2. 3 Oenococcus oeni
•

Description: Oenococcus oeni is a gram-positive, non-motile, chemoorganotrophic lactic
acid bacterium that grows in chains of circular to ellipsoidal cells.
• Growth Conditions: The optimum temperature for growth of Oenococcus oeni is 30°C.
• Nutritional Requirements
Carbon source: Most strains of O. oeni utilize L-arabinose, fructose, and ribose
but not galactose, lactose, maltose, melezitose, raffi nose, or xylose. O. oeni can
metabolize malic acid found in grapes to form lactic acid through malolactic
fermentation.
 Nitrogen source: O. oeni can assimilate nitrogenous substances in the form of
amino acids and peptide but not inorganic nitrogen-containing compounds.


25