Adrian Gauna

-Ingeniero en Alimentos (UNLu - Argentina)

-Master en Ciencias y Tecnología de Alimentos (UNL - Argentina)
-Especialista en Quesería Internacional (ENILBIO-Poligny-Francia)
39 años de experiencia en el sector lácteo.

-Director Regional de Sacco System con sede en Italia, empresa

para la que trabajo desde hace 30 años.

-Región geográfica donde desempeňa su actividad laboral :

América del Norte, América Central y América del Sur, Francia,

Espaňa, Portugal, Benelux, Norte y Centro de Africa, Israel, Australia
y NZ. (Inglaterra, Irlanda, Escocia 2004 a 2010 y Cina 2016 a 2019)

Sacco System | saccosystem.com

 M. Sc. Adrian Gauna
Regional Director Sacco System
1/3 of the world food is wasted
Preservation in opposition to deterioration
To limit the deterioration you can act on the process or on the ingredients

• Pasteurization
• Sterilization
• UV treatment

Process
Natural antimicrobials

Cultures with protective effect

Sorbates
Benzoates
Propionate
Ingredients Nisin
Natamycin
Artificial antimicrobials
Cultures with protective effect: how do they work?

Reducing food waste by prolonging the shelf life of foods and keeping them fresh and safe for a longer period

Cultures with protective effect function through multiple biological interactions with the food matrix and with the spoilage
microrganisms present in the food;
The protective effect of fermentation is the result of three main interacting mechanisms:
 Produce
inhibitory
molecules
(metabolites, e.g.,
bacteriocins, organic
acids, peptides)

Siedler et al., 2019

 Food Cultures on top of good manufacturing principles
Food cultures add an extra hurdle to prevent the growth of unwanted microorganisms and maintain the quality and safety
of the food

Additional hurdle to protect the built-in  Control of microflora

quality and food safety throughout the
shelf life of the product
Food  Stable product quality
Culture  Improved microbial safety

General HACCP/GMP Additional hurdles:

management to ensure temperature, pH, aw
the best possible quality packaging, etc.
and food safety

Selection of raw materials -

optimization of process/recipe

HACCP - Good Manufacturing Practice

 An example of how food cultures can reduce food waste by 30%
Adding selected food cultures during the production of yogurt prolongs freshness and extends the shelf life by 7 days.

• Every year, up to 17% of all yogurt produced in the EU is wasted, which equals a total of 1.5 million tons of yogurt.

• Food cultures can delay the spoilage of fermented dairy products like yogurt, which can potentially reduce yogurt
waste in the EU by up to 440,000 tons, which equals a 30% waste reduction.
Sacco’s range of cultures with protective effect

Anti Anti Pseudomonas

Anti Heterofermentative &
Anti Clostridia Anti Listeria
Yeasts & Moulds & Anti-psychrotrophic
Anti-Coliforms bacterias
How Sacco can help their clients  R&D microbiology lab
The best solution for each problem

CONTAMINATION ISOLATION & IDENTIFICATION

IN VITRO AND MODEL INHIBITION TESTS
OF SPOILAGE ORGANISMS

…some examples
BEST SOLUTION
Case of study: Turkish cheese factory
Problems of yeast and mould contamination in Ayran and white cheese
ISOLATION & IDENTIFICATION

Rhodotorula spp.
P. crostosum
P. commune/biforme
P. spinuloramigenum/
glabrum
CONTAMINATION Didymella spp. In vitro TEST
Rhodotorula spp Penicillium commune/ biforme

BEST SOLUTION

LPRA, LR4PD,
LPE, LPRF,
CPR4P01
Case of study: Chilean Gouda cheese
Results: 10 DOSES/100L

LPAL, SP1 & LRB showed the

strongest effect against the
yeasts isolates –
dose-dependent effect
 Compete for Anti – Yeast and Moulds
nutrients Fermented milk products
(e.g., Mn depletion, a.a.
consumption)

Produce
inhibitory
molecules
(metabolites, e.g., organic
acids, diacetyl, peptides)
Model systems for further validations

3ml in each well

Mini yogurt-model
SYAB 1
SYAB 1

SYAB 1
+ 5% Bromocresol green/purple +
Incubation at
LPRA 10°C for 42 days
SYAB 1
+
SYAB 1 + *colture with LR4PD
protective effect
1 CFU/ml 20 CFU/ml
G. candidum G. candidum

42 °C until pH 4,8
and cooling at 22°C
x 30 min

Moulds plate counts demonstrated that

protective culture contained the spoilage mould!
Effect of volatile compounds

Produce
inhibitory
molecules
Diacetyl
Know your enemy: yeasts
Anti-yeasts activity screening results
Know your enemy: moulds
Anti-moulds activity screening results
Synergistic activity of LAB
 Fermented cream
XXXX
Fermented cream

LPRA showed
effects against all
3 isolates both at
1 and 10 doses
 Yogurt

YYY
 Yogurt

Inhibitory activity against Penicillium bialowiezense

Strong inhibition
when using 5 doses

Inhib. sporulation > inhib. mycelia

Case of study: Belgian cheese factory
Geothricum candidum is the main contaminant in this factory
ISOLATION & IDENTIFICATION

Geothricum candidum

CONTAMINATION In vitro TEST

BEST SOLUTION

LPRA, LR4PD,
LPE, LPRF, SP 1,
CLPC
Lacticaseibacillus paracasei BGP 1 vs Heterofermentative & Propionibacteria

Without BGP 1 With BGP 1 Without BGP 1 With BGP 1

Anti
Heterofermentative
& Propionibacteria
and
Anti-coliform

BGP 1 activity vs Lb. heterofermentative, BGP 1 does not inhibit selected PAB flora
unwanted PAB and Clostridia development such as PB 1
Lyofast CPR4P1 – Queso Fresco elaborado con leche cruda

T1 M2 T2 M2

Testigo 30 dosis/1000lts
(sin cultivo CPR4P1
CPR4P1 – Queso Fresco elaborado con leche pasteurizada
PRUEBAS CON CPR4P1
Pruebas en leche pasteurizada
Fecha: 1-6-21
Queso fresco (sin cultivos lácticos)

Codificación de muestras
Dosis/1000 LTS
T1 50
T2 30
T3 sin nada

LECHE ANTES DE CUAJAR SALIDA DE PRENSA* A LAS 72 HORAS

Id. Muestra C. totales E.coli C. totales E.coli C. totales E.coli pH
ufc/mL ufc/mL ufc/g ufc/g ufc/g ufc/g

T1 <10 <10 <10 <10 4 x 10exp3 <10 6,55

T2 <10 <10 <10 <10 3,2 x 10exp4 <10 6,53
T3 <10 <10 3 x 10exp2 <10 1,1 x 10exp5 <10 6,56
LR B reduces the growth of unwanted indigenous microorganism in milk
Improvement of milk storage stability and quality

Psychrotrophic bacteria growth in milk stored at Effect of LR B on psychrotrophic bacteria in milk

4°C MILK maturation (48 hrs) MILK

5,80 MILK+ LRB 1,00E+07 MILK+ LRB

5,60
5,40
5,20
LOG CFU/ml

1,00E+06
5,00

CFU/ml
4,80 - 90% - 72%
4,60 - 97%
Anti Pseudomonas & 4,40
4,20
1,00E+05
- 97% - 88% - 88%

Psychrotrophic 4,00
bacteria T0 T24 T48
1,00E+04
Time (hrs) Trial 1 Trial 2 Trial 3 Trial 4 Trial 5 Trial 6

LR B keeps the overall quality of your milk steady.

 Focus on Milk protein quality (addition of 1,15 ml of KH2PO4)
By adding culture with protective effect milk protein quality is preserved compared with control

Psychrophila 10^3 CFU/ml Psychrophila 10^5 CFU/ml

1 1
Milk quality score

Milk quality score

0,5 0,5

0 0
3hrs 5hrs 12 hrs 3hrs 5hrs 12 hrs 3hrs 5hrs 12 hrs 3hrs 5hrs 12 hrs 3hrs 5hrs 12 hrs 3hrs 5hrs 12 hrs
LRB 0,5 D/100L CNB AP 0,5 D/100L CNB AP + LRB 1 D/100L LRB 0,5 D/100L CNB AP 0,5 D/100L CNB AP + LRB 1 D/100L

Control T24 T48 T72 Control T24 T48 T72

The quality of the milk is always higher when PC are added

Milk Pasteurization leads to strain inactivation
After pasteurization LRB count is < 2 log CFU/ml (limit of detection 100 CFU/ml)

LRB after 24hrs of incubation at 4°C LRB after 48hrs of incubation at 4°C
5,50 5,50

5,00 5,00

4,50 4,50

Log CFU/ml
Log CFU/ml

4,00 4,00

3,50 3,50

3,00 3,00

2,50 2,50
<2 <2 <2 <2
2,00 2,00
5D/10ton 10D/10 ton 5D/10ton 10D/10 ton
T0 T24 4°C T24 past T0 T48 4°C T48 past
CNB AP controls the development of Pseudomonas in mozzarella cheese
CNB AP mantains Pseudomonas contamination under the
discoloration level
9,0
Control inoculated with.
8,0 Discoloration level Pseudomonas
7,0
6,0
Log CFU/g
5,0
4,0
3,0
2,0
1,0
0,0
Negative control + CNB AP
T0 T11 T20
Anti Pseudomonas &
Psychrotrophic Reduction of Pseudomonas development in Mozzarella
bacteria 100%
90%
80% Sample inoculated with
70% Pseudomonas and CNB AP
60%
50%
40%
30%
20% More than 90% of inhibition of
10%
0% Pseudomonas by using CNB AP
Positive control +CNB AP Positive control +CNB AP
% of contamination level
T11 T20
Data collected after 11 and 20 days of incubation at 4°C
Selection of new strains for a new blend active vs Clostridia (on going project)
Strains were tested vs Clostridia isolated from ripened cheese

Lacticaseibacillus rhamnosus
Assessment of the
genetic diversity
Lacticaseibacillus paracasei

Lb. paracasei

Anti Clostridia
In vitro test vs.
Clostridia isolated
from cheeses

Lb. rhamnosus
CNB AL vs Listeria spp. and Listeria monocytogenes on Gorgonzola rind
800 wheels of cheese were treated during the sperimentation

Legend:
1-3-5-7: not treated samples
2-4-6-8: treated samples

Anti Listeria
CNB AL was able to control the
development of Listeria
monocytogenes in treated samples
comparing to the non treated one
<1
(- 3 log CFU/g)
<1 <1 <1
Technological characterization of Lyofast CNB AP

CNB AP growth at low temperatures 22% NaCl survival

10,00 8,00

9,50 7,90
5°C 7°C 10°C 13°C
+ 2 Log CFU/ml in + 3 Log CFU/ml in + 3 Log CFU/ml in + 3 Log CFU/ml in
9,00 7,80
72 hrs 72 hrs 48 hrs 48 hrs; 4 Log
CFU/ml in 72 hrs
8,50
7,70

8,00
7,60
Log CFU/ml

Log CFU/ml
7,50
7,50

7,00
7,40
6,50
7,30
6,00
7,20
5,50
7,10
5,00
T0 T6 T12 T48 T72 T0 T6 T12 T48 T72 T0 T6 T12 T48 T72 T0 T6 T12 T48 T72
7,00
5°C 7°C 10°C 13°C C. divergens C. maltaromaticum

C. divergens C. maltaromaticum T0 hrs T7 hrs

Data collected after 7 hrs of incubation at 4°C

 Pseudomonas spp.
Main responsible of spoilage in fresh products and of discoloration defects in Mozzarella cheese

- Ubiquitous and psychrotolerant Gram-negative bacteria, frequently isolated

from refrigerated raw milk;
- In general, post-pasteurization contamination in dairy plants, often in
Mozzarella storage liquid or wastewater;
- Main responsible of spoilage in fresh products, at low temperatures;
- Can produce biofilm;
- Different strains and species associated with discoloration defects in fresh
cheeses expecially in Mozzarella  some strains produce pigments
(pyoverdine or pyocianin) at a certain load;
- Growth till 107 CFU/g negatively affect:
• the sensory properties of the product ;
• the appearance, unwanted discoloration possible.
References
• Quintieri L, Caputo L, Brasca M, Fanelli F. (2021) Recent Advances in the Mechanisms and Regulation of QS in Dairy Spoilage by Pseudomonas spp. Foods.; 10(12):3088.
• del Olmo, A., Calzada, J., & Nuñez, M. (2018). The blue discoloration of fresh cheeses: A worldwide defect associated to specific contamination by Pseudomonas fluorescens. Food Control, 86, 359-366.
• Kumar, H., Franzetti, L., Kaushal, A., & Kumar, D. (2019). Pseudomonas fluorescens: A potential food spoiler and challenges and advances in its detection. Annals of Microbiology, 69(9), 873-883.
Lyofast CNB AP
Blend of C. divergens and C. mataromaticum used to improve the shelf life of chilled food

- Gram positive rod shaped lactic acid bacteria (LAB) isolated from different ecological
niches;

- Bacteriocins producers  small peptide inhibiting the growth of specific targets;

- Lyofast CNB AP, due to similar growth conditions (pH and temperature variation) of
psychotropic spoilage and pathogen microorganisms, can improve the durability of
chilled foods by reducing or inhibiting the growth of Pseudomonas spp.

References
• Elsser-Gravesen, D., Elsser-Gravesen, A., 2013. Biopreservatives. In: Biotechnology of Food and Feed Additives. Springer, Berlin Heidelberg, pp. 29-49..
• Leisner, J.J., Laursen, B.G., Prevost, H., Drider, D., Dalgaard, P., 2007. Carnobacterium: positive and negative effects in the environment and in foods. FEMS Microbiol.
Rev. 31, 592e613.
• Afzal, M.I., Jacquet, T., Delaunay, S., Borges, F., Milliere, J.B., Revol-Junelles, A.M., Cailliez-Grimal, C., 2010. Carnobacterium maltaromaticum: identification, isolation
tools, ecology and technological aspects in dairy products. Food Microbiol. 27 (5), 573e579..
Experimental design
Validation of Mozzarella cheese brine model (preliminary screening was carried out by Università Cattolica
del Sacro Cuore)

Trained panel

Created with BioRender.com

 Pseudomonas spp. cocktail
Most of the Pseudomonas spp. used were isolated from mozzarella cheese
Species Isolation origin

Pseudomonas putida Mozzarella cheese

Pseudomonas chlororaphis Mozzarella cheese

Pseudomonas aeruginosa Collection strain (provided by LGC standard)

Pseudomonas fluorescens Mozzarella cheese

Pseudomonas fluorescens Mozzarella cheese

Pseudomonas jessenii Mozzarella cheese

Pseudomonas koreensis Mozzarella cheese

Pseudomonas luteola Water

Pseudomonas lactis Mozzarella cheese (Kindly provided by CNR ISPA, Bari)

Pseudomonas lactis Mozzarella cheese (Kindly provided by CNR ISPA, Bari)

All these strains were used in equal part (v/v) to directly inoculate mozzarella brine (called «Pseudomonas cocktail»)
 Lyofast CNB AP mantains Pseudomonas contamination under the
discoloration level
Control inoculated with
9,0 Pseudomonas
8,0
Discoloration level
7,0

6,0
Log CFU/g

5,0

4,0

3,0

2,0

1,0

0,0
Negative control + Lyofast CNB AP
Sample inoculated with
Pseudomonas and Lyofast CNB AP
T0 T11 T20

Data collected after 11 and 20 days of incubation at 4°C

Ref: Carminati, D., Bonvini, B., Rossetti, L., Zago, M., Tidona, F., & Giraffa, G. (2019). Investigation on the presence of blue pigment-producing Pseudomonas strains along a production line of fresh
mozzarella cheese. Food Control, 100, 321-328.
Lyofast CNB AP reduces the colonization of Pseudomonas cocktail in Mozzarella

100%

90%

80%

70%
% of colonization

60%

50%

40%

30%

20%

10%

0%
Positive control +Lyofast CNB AP Positive control + Lyofast CNB AP
T11 T20

T11 Positive control T11 +Lyofast CNB AP T20 Positive control T20 + Lyofast CNB AP

Data collected after 11 and 20 days of incubation at 4°C

The addition of Lyofast CNB AP do not cause off-flavour in Mozzarella
Data collected after 20 days of incubation at 4°C. Trained panel.

Sensory evaluation of Mozzarella samples

non treated sample + Lyofast CNB AP
Surface colour
Rancid 7 Brightness
Oxydized Surface homogeneity
Fermented 6 Interior color
Cooked milk 5 Structural homogeneity
4
Yoghurt Syneresis
3
Butter 2 Olfactory intensity

Floral/Fruity 1 Positive Flavour

0
Vanilla Off-flavour

Fresh milk Crumbliness

Trigeminal Adhesiviness
Umami (Sapidity) Oiliness
Salty Deformability
Bitter Rubberiness
Acidic Hardness
Sweet
Lyofast CNB AP inhibits the productions of pigments by Pseudomonas
lactis (Ps. lactis provided by CNR ISPA, Bari)
8,00
Discoloration level
7,50

7,00

6,50

6,00
LOG CFU/ml

5,50

5,00

4,50

4,00

3,50

3,00
Pseudomonas lactis Ps. lactis + Lyofast CNB AP
T0 T11 T20
 Experimental design
Utilization of Ricotta cheese model (the screening was carried out by Università di Sassari)

Created with BioRender.com

Ref: Spanu C, Piras F, Mocci AM, Nieddu G, De Santis EPL, Scarano C. Use of Carnobacterium spp protective culture in MAP packed Ricotta fresca cheese to control Pseudomonas spp. Food Microbiol. 2018 Sep;74:50-56
Containment of Pseudomonas fluorescens develpoment in Ricotta
cheese by Lyofast CNB AP
Pseudomonas fluorescens count
10,00

9,00

8,00

7,00

6,00
Log CFU/g

5,00

4,00

3,00

2,00

1,00

0,00
Negative control + Lyofast CNB AP

T0 T7 T14 T21

Data collected after 7, 14 and 21 days of incubation at 4°C

Ref: Spanu C, Piras F, Mocci AM, Nieddu G, De Santis EPL, Scarano C. Use of Carnobacterium spp protective culture in MAP packed Ricotta fresca cheese to control Pseudomonas spp. Food
Microbiol. 2018 Sep;74:50-56
Lyofast CNB AP reduces the colonization of Pseudomonas fluorescens in
Ricotta cheese after 21 days
100%

90%

80%

70%
% of colonization

60%

50%

40%

30%

20%

10%

0%
T7 T14 T21
Negative control + Lyofast CNB AP

Data collected after 7, 14 and 21 days of incubation at 4°C

Ref: Spanu C, Piras F, Mocci AM, Nieddu G, De Santis EPL, Scarano C. Use of Carnobacterium spp protective culture in MAP packed Ricotta fresca cheese to control Pseudomonas spp. Food
Microbiol. 2018 Sep;74:50-56
MOZZARELLA/PIZZA CHEESE/PASTA FILATA Technological characterization of CNB AP
Survival to stretching gradient

Lyofast CNB AP*

Created with BioRender.com

*Lyofast CNB AP: 5D/100L or 10D/100L; Brine recipe: 0,6% NaCl, 1% of a 35% solution of CaCl2, 0,1% of lactose (to mimic the lactose release from mozzarella cheese).
Lyofast CNB AP can survive to stretching gradient
Survival and development of CNBAP after stretching gradient
10,00

9,00

8,00
Log CFU/ml

7,00

6,00

5,00

4,00

3,00
T0 after treatment after 11 days of incubation T0 after treatment after 11 days of incubation
at 10°C at 10°C
MRS BRINE
5D/100l CNBAP 10D/100l CNBAP
Lyofast CNB AP can control Pseudomonas development after stretching gradient

100%

90%

80%

70%
% of colonization

60%

50%

40%

30%

20%

10%

0%
Positive control 5D/100l Lyofast CNB AP 10D/100l Lyofast CNB AP 5D/100l Lyofast CNB AP 10D/100l Lyofast CNB AP
Not treated Treated
% of contamination MRS % of contamination BRINE
Technological characterization of CNB AP

Survival to different concentrations of potassium sorbate

Created with BioRender.com

Evaluation of Lyofast CNB AP growth in presence of potassium sorbate

Growth of Lyofast CNB AP in presence of potassium sorbate

2,5

1,5
OD600

0,5

0
Lyofast CNB AP Lyofast CNB AP + 0,5 g/kg K sorbate Lyofast CNB AP + 1,0 g/kg K sorbate Lyofast CNB AP + 2,0 g/kg K sorbate

T0 hrs T24 hrs

Effectiveness of Lyofast CNB AP stored at T > 4°C before the utilization in
brine
Lyofast CNB AP load is not affected by incubation at 18°C x 24 hrs

7,50

7,40

7,30
LOG CFU/ml

7,20

7,10

7,00

T0 T24
Lyofast CNB AP can control Pseudomonas development after 24 hrs at
18°C
100%

90%

80%

70%
% of colonization

60%

50%

40%

30%

20%

10%

0%
T0 T11 T20 T0 T11 T20
Standard conditions Experimental conditions
Best practices of Lyofast CNB AP application

- The sperimentation done showed the Lyofast CNB AP effectiveness vs Pseudomonas in laboratory
conditions by inoculating CNB AP directly in Mozzarella brine;

- The validation of Lyofast CNB AP in dairy plants requires to apply the following conditions:
- Inoculum (the dosage has to be defined with the reference technologist) of Lyofast CNB AP directly in
Mozzarella brine packaged in thermo-sealed trays;

- The conservation has to be done at 4°C in optimal conditions;

- Different applications (such as Burrata, Pizza cheese, Cherry Mozzarella) would require a specific
validation: laboratory tests would allow the definition of the best conditions to be applied.
Contact:

M. Sc. Adrian Gauna

Mobile :+39 3356700657
Email: [email protected]