# WP2: Innovative plant-based dairy analogues: Fermented products

Gwénaël Jan, Valérie Gagnaire, Charles Silande

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## To cite this version:

Gwénaël Jan, Valérie Gagnaire, Charles Silande. WP2: Innovative plant-based dairy analogues: Fermented products. LocalNutLeg, UMR STLO INRAE, May 2023, Rennes, France. hal-04090153

HAL Id: hal-04090153

https://hal.inrae.fr/hal-04090153

Submitted on 5 May 2023

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Distributed under a Creative Commons Attribution 4.0 International License
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LOCALNUTLEG

WP2 : Innovative plant-based dairy
analogues :
Fermented products

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LOCALNUTLEG
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<figure><img src="image_6.png" /><figcaption>gwenael.jan@inrae.fr</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>valerie.gagnaire@inrae.fr</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>charles.silande@inrae.fr</figcaption></figure>

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# Task 2.2. Fermented probiotic plant-based beverages

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# Task 2.2. Fermented probiotic plant-based beverages

## Experimental strategy :

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## Lactic Acid Bacteria (LAB)

Gram +
Cocci or bacilli

**Feature :** Acid lactic production
from different carbohydrates

**Functions :** Acidification,
coagulation, aromas/flavours,
proteolysis
Used in all fermented dairy
products (cheese, yogurt,
butter...)

**Examples of LAB:**

* Streptococcus thermophilus

* Lactococcus lactis

* Lactobacillus delbrueckii subsp. bulgaricus

* Lacticaseibacillus casei

<figure><img src="image_16.png" /><figcaption>S. thermophilus</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>L. delbrueckii subsp. bulgaricus</figcaption></figure>

<figure><img src="image_18.png" /></figure>

**In the WP2 Localnutleg :**
Use for the fermented
milk, the yogurt-type and
the cheese analogue

## Propionic Acid Bacteria (PAB)

Gram +
Cocobacilli (Short bacilli)

**Feature :** Propionic acid (involved in the test and smell of cheese) production from acid lactic

**Functions :** Aromas/flavours
Texture (Swiss-type cheese hole)
Used in cheeses

**Examples of PAB:**

* Propionibacterium freudenreichii

* Acidipropionibacterium acidipropionici

<figure><img src="image_19.png" /><figcaption>P. freudenreichii</figcaption></figure>

<figure><img src="image_20.png" /><figcaption>A. acidipropionici</figcaption></figure>

<figure><img src="image_21.png" /></figure>

**In the WP2 Localnutleg :**
Use for the cheese
analogue with the LAB
## Strains pre-selection

Pre-selection of 38 strains :

- 23 LAB

- 15 PAB

### Propionic Acid Bacteria (PAB)

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### Lactic Acid Bacteria (LAB)

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# Task 2.2. Fermented probiotic plant-based beverages

## Screening of the pre-selected lactic acid bacteria strains

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Some strains give a lactic (yogurt) note, promising for fermented milk making

<table><thead><tr><th>Species</th><th>Strain number</th><th>pH drop</th><th>Organoleptic assessment (appreciation converted into ratings)</th><th>Anti-inflammatory properties (IL10/IL12)</th></tr></thead><tbody><tr><td>Lactococcus lactis subsp. lactis</td><td>Lla NCDO2118</td><td>2,74</td><td>8</td><td>8</td></tr><tr><td>Lactococcus lactis subsp. lactis</td><td>Lla CIRM-BIA 242</td><td>2,42</td><td>6</td><td>0,02</td></tr><tr><td>Lactococcus lactis subsp. lactis</td><td>Lla Bioprox 5676</td><td>2,61</td><td>7</td><td>0,06</td></tr><tr><td>Lactococcus lactis subsp. cremoris</td><td>Llc MG1363</td><td>1,55</td><td>3</td><td>8</td></tr><tr><td>Lactiplantibacillus plantarum</td><td>Lp CIRM-BIA1420</td><td>2,55</td><td>2</td><td>6,58</td></tr><tr><td>Lactiplantibacillus plantarum</td><td>Lp CIRM-BIA2107</td><td>2,57</td><td>2</td><td>11,18</td></tr><tr><td>Lactiplantibacillus plantarum</td><td>Lp Bioprox 1096</td><td>2,49</td><td>2</td><td>12,53</td></tr><tr><td>Lacticaseibacillus casei</td><td>Lc CIRM-BIA1643</td><td>1,04</td><td>7</td><td>Well-known probiotic effect</td></tr><tr><td>Lactobacillus delbrueckii subsp. lactis</td><td>Ldl CIRM-BIA207</td><td>1,39</td><td>4</td><td>11,75</td></tr><tr><td>Lactobacillus delbrueckii subsp. lactis</td><td>Ldl CIRM-BIA303</td><td>1,32</td><td>4</td><td>31,97</td></tr><tr><td>Lactobacillus johnsonii</td><td>Lj Bioprox 5467</td><td>2,52</td><td>4</td><td>Inconnu</td></tr><tr><td>Streptococcus salivarius subsp. thermophilus</td><td>St CIRM-BIA772</td><td>1,5</td><td>10</td><td>1,5</td></tr><tr><td>Streptococcus salivarius subsp. thermophilus</td><td>St CIRM-BIA251</td><td>2,88</td><td>9</td><td>0,1</td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>Ldb CIRM-BIA905</td><td>0,46</td><td>8</td><td>71,98</td></tr><tr><td>Limosilactobacillus fermentum</td><td>Lf CIRM-BIA697</td><td>0,31</td><td>5</td><td>17,5</td></tr><tr><td>Limosilactobacillus fermentum</td><td>Lf CIRM-BIA2156</td><td>0,09</td><td>5</td><td>5,27</td></tr><tr><td>Lacticaseibacillus rhamnosus</td><td>Lr CIRM-BIA1107</td><td>0,98</td><td>7</td><td>5,86</td></tr><tr><td>Lacticaseibacillus rhamnosus</td><td>Lr GG</td><td>0,79</td><td>4</td><td>4</td></tr><tr><td>Lactobacillus helveticus</td><td>Lh CIRM-BIA100</td><td>0,38</td><td>5</td><td>100</td></tr><tr><td>Lactobacillus helveticus</td><td>Lh CIRM-BIA103</td><td>0,42</td><td>5</td><td>65,12</td></tr><tr><td>Lactobacillus acidophilus</td><td>La NCFM</td><td>0,86</td><td>3</td><td>11</td></tr><tr><td>Lactobacillus acidophilus</td><td>La CIRM-BIA444</td><td>2,05</td><td>5</td><td>0,29</td></tr><tr><td>Bifidobacterium longum</td><td>BI Bb536</td><td>0,66</td><td>8</td><td>10</td></tr></tbody></table>

### Selection of strains to design consortia :

#### Mesophilic consortium (30°C) :

- Lactococcus lactis subsp. lactis NCDO2118

- Lacticaseibacillus casei CIRM-BIA1643

#### Thermophilic consortium (43°C) :

- Streptococcus thermophilus CIRM-BIA772

- Lactobacillus delbrueckii subsp. bulgaricus CIRM-BIA905

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# Task 2.2. Fermented probiotic plant-based beverages

## Screening of the pre-selected propionic acid bacteria strains

<table><thead><tr><th>Species</th><th>Strain number</th><th>pH drop</th><th>Organoleptic assessment (appreciation converted into ratings)</th><th>Anti-inflammatory properties (IL10/IL12)</th></tr></thead><tbody><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1652</td><td>2,21</td><td>3</td><td>52,24</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1766</td><td>2,39</td><td>3</td><td>53,91</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1767</td><td>1,74</td><td>3</td><td>79,32</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1768</td><td>2,12</td><td>3</td><td>61,88</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1994</td><td>1,7</td><td>3</td><td>75,82</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1995</td><td>1,57</td><td>5</td><td>68,49</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1998</td><td>2,17</td><td>3</td><td>77,57</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA2003</td><td>1,81</td><td>3</td><td>44,98</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA1424</td><td>0,95</td><td>5</td><td>84,46</td></tr><tr><td>Acidipropionibacterium acidipropionici</td><td>Aa CIRM-BIA2000</td><td>1,55</td><td>5</td><td>71,05</td></tr><tr><td>Propionibacterium freudenreichii</td><td>Pf CIRM-BIA120</td><td>0,77</td><td>8</td><td>19,97404906</td></tr><tr><td>Propionibacterium freudenreichii</td><td>Pf CIRM-BIA141</td><td>0,74</td><td>5</td><td>45,86368605</td></tr><tr><td>Propionibacterium freudenreichii</td><td>Pf CIRM-BIA129</td><td>0,7</td><td>5</td><td>45,62</td></tr><tr><td>Propionibacterium freudenreichii</td><td>Pf CIRM-BIA1466</td><td>1,15</td><td>5</td><td>39,88066655</td></tr><tr><td>Propionibacterium freudenreichii</td><td>Pf CIRM-BIA1472</td><td>0,72</td><td>8</td><td>34,60363517</td></tr></tbody></table>

Meat notes

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Cheesy notes

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Promising for vegetable cheese making

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# Task 2.2. Fermented probiotic plant-based beverages

## Creation and testing of consortia with the selected strains

<table><thead><tr><th>Code</th><th>Species</th><th>Strain number</th><th>pH drop</th><th>Organoleptic assessment (appreciation converted into ratings)</th></tr></thead><tbody><tr><td rowspan="2">Mesophilic consortium :<br>St 772 + Ldb 905</td><td>Streptococcus thermophilus</td><td>St CIRM-IA772</td><td rowspan="2">2,65</td><td rowspan="2">4</td></tr><tr><td>Lactobacillus delbrueckii subsp. bulgaricus</td><td>Ldb CIRM-BIA905</td></tr><tr><td rowspan="2">Thermophilic consortium :<br>Lla NCDO2118 + Lc 1642</td><td>Lactococcus lactis subsp. lactis</td><td>Lla NCDO2118</td><td rowspan="2">2,4</td><td rowspan="2">3,00</td></tr><tr><td>Lacticaseibacillus casei</td><td>Lc CIRM-PB1643</td></tr></tbody></table>

Strains can give good flavours alone but once in consortium give bad ones

rancid and fatty notes

Lactic and fresh notes

Selection of two consortium for the following study :

- LAB consortium

- LAB+PAB consortium (addition of a PAB strain (Pf CIRM-141)) to improve the probiotic effect

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# Task 2.2. Fermented probiotic plant-based beverages

## Growth kinetics – pH results

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*S. thermophilus* is responsible for pH drop

Similar results with 2,5% sucrose
Slightly stronger acidification
(Final pH of 5,2)

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# Task 2.2. Fermented probiotic plant-based beverages

## Growth kinetics – bacterial count results

Growth of S. thermophilus (1 log)
Lower growth in the LAB+PAB
consortium (competition for free
amino acids)

<figure><img src="image_35.png" /><figcaption>Strain population at 37°C as function of time in almond milk La Mandorle</figcaption></figure>

Population of L. casei needed to
induce a probiotic effect : 10⁸
CFU/ml

Slight growth of L. casei in both
consortia (use of
fructose/glucose provided by S.
thermophilus which degrades
sucrose)

Population of P. freudenreichii
stable (10⁸ CFU/ml)

Enough to induce a probiotic
effect

<figure><img src="image_36.png" /></figure>
# Task 2.2. Fermented probiotic plant-based beverages

## Growth kinetics – carbohydrates consumption

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<figure><img src="image_42.png" /><figcaption>Sucrose concentration in almond milk before and after fermentation</figcaption></figure>

<table><caption>Sucrose concentration in almond milk with 2,5% sucrose before and after fermentation</caption><thead><tr><th></th><th>Sucrose concentration in g/L</th><th>Time in hours</th></tr></thead><tbody><tr><td>Concentration 0h</td><td>30</td><td>0h</td></tr><tr><td>S. thermophilus 772</td><td>25</td><td>24h</td></tr><tr><td>L. casei 1643</td><td>20</td><td>24h</td></tr><tr><td>consortium LAB</td><td>15</td><td>24h</td></tr><tr><td>consortium LAB+PAB</td><td>10</td><td>24h</td></tr></tbody></table>

<table><thead><tr><th>Sucrose concentration in g/L</th><th>Time in hours</th></tr></thead><tbody><tr><td>Concentration 0h</td><td>30</td></tr><tr><td>S. thermophilus 772</td><td>25</td></tr><tr><td>L. casei 1643</td><td>20</td></tr><tr><td>consortium LAB</td><td>15</td></tr><tr><td>consortium LAB+PAB</td><td>10</td></tr></tbody></table>

No consumption of the sucrose by L. casei (sucrose -)
Consumption of all the sucrose (2 g/L) by S. thermophilus (sucrose +)

Consumption of sucrose is slightly more important (3 g/L) in almond milk with 25 g/L sucrose
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# Task 2.2. Fermented probiotic plant-based beverages

## Growth kinetics – carbohydrates consumption

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<figure><img src="image_50.png" /><figcaption>Fructose concentration in almond milk before and after fermentation</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>Fructose concentration in almond milk with 2,5% sucrose before and after fermentation</figcaption></figure>

<figure><img src="image_52.png" /></figure>

Fructose release by *S. thermophilus* (sucrose + and fructose +/-)

Fructose consumption by *L. casei* (fructose +)

Same conclusion but with more fructose released (because more sucrose were used by *S. thermophilus*)
<figure><img src="image_53.png" /></figure>

# Task 2.2. Fermented probiotic plant-based beverages

## Growth kinetics – carbohydrates consumption

<figure><img src="image_54.png" /></figure>

<figure><img src="image_55.png" /><figcaption>Glucose concentration in almond milk before and after fermentation</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>Glucose concentration in almond milk with 2,5% sucrose before and after fermentation</figcaption></figure>

<figure><img src="image_57.png" /></figure>

Glucose consumption by all strains (glucose +)
Residual glucose because S. thermophilus releases some (sucrose +)

Same conclusion but S. thermophilus metabolizes more sucrose than it consumes glucose
<figure><img src="image_58.png" /><figcaption>Sucrose and organic acid concentrations for LAB+PAB consortium</figcaption></figure>

<figure><img src="image_59.png" /></figure>

Sucrose consumption and lactate production by LAB

Sucrose consumption and lactate production by LAB
Lactate consumption and propionate/acetate production by PAB
# Task 2.2. Fermented probiotic plant-based beverages

## Growth kinetics – Amino acids

<figure><img src="image_60.png" /></figure>

Very low amino acid released

High consumption of free amino acids

⇒ The strains are not proteolytic and used the free amino acids in almond milk (to be confirmed with an SDS-PAGE gel and OPA assay)

⇒ Could explain the limited growth of *S. thermophilus* in the presence PAB
(competition for free amino acids)

⇒ Same results with or without 2.5% sucrose

<figure><img src="image_61.png" /></figure>
# Task 2.2. Fermented probiotic plant-based beverages

## Sensory analysis perform in WP5

### Main result of the sensory analysis

Dimensions (axis F1 and F2: 85.29 %)

<figure><img src="image_62.png" /></figure>

### PCA of the sensory analysis perform in WP5

Almond Milk

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Almond milk with
2,5% sucrose added

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Consortium
LAB

Foaminess
Astringent
Granularity
Roughness

Sweet
Yogurt
Slip in mouth

Consortium
LAB+PAB

Cheese
Intensity
Acid
Bitter
Granularity

Foaminess
Astringent
Granularity
Roughness

<figure><img src="image_65.png" /><figcaption>Fermented milk
before shaking</figcaption></figure>

<figure><img src="image_66.png" /></figure>

<figure><img src="image_67.png" /><figcaption>Fermented milk
after shaking</figcaption></figure>

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## Task 2.3. Yogurt-type fermented probiotic gels

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>
<figure><img src="image_72.png" /></figure>

# Task 2.3. Yogurt-type fermented probiotic gels

## Experimental strategy

Need a higher protein concentration to obtain a firmer gel

2 methods explored :

- Enrichment of almond milk with powder

- Use of protein-enriched plant-based milk supplied by Fraunhofer

### Almond/chickpea milk mixes

<table><thead><tr><td>Almond</td><td>Chickpea</td></tr></thead><tbody><tr><td>100%</td><td>0%</td></tr><tr><td>75%</td><td>25%</td></tr><tr><td>50%</td><td>50%</td></tr><tr><td>25%</td><td>75%</td></tr><tr><td>0%</td><td>100%</td></tr></tbody></table>

<figure><img src="image_73.png" /></figure>

Enrichment of almond milk with powder (target of 4% of protein)

<figure><img src="image_74.png" /></figure>

ChickP

- Use of multiple powders (fatty and defatted almond flour, almond powder, ChickP powder (G910 & S930))

- Heat treatment to sterilize the mixture (115°C – 20 min)

Use of protein-enriched plant-based milk (mix almond/chickpea) supplied by Fraunhofer (≈3% of protein)

<figure><img src="image_75.png" /></figure>

Fraunhofer

Fermentation of these plant-based milk enriched in protein with the S. thermophilus previously selected

- Growth assessment (pH, bacterial count)

- Organoleptic properties assessment (lab test (5-6 tasters))

Study of selected strains on Fraunhofer's milks (in discussion)

- Addition of a probiotic bacteria (L. casei or L. delbrueckii subsp. bulgaricus)

- Overall composition of the milks and target metabolites (carbohydrates, organic acid...)

- Rheological measurements

- Sensory analysis

<figure><img src="image_76.png" /></figure>
# Task 2.3. Yogurt-type fermented probiotic gels

## Enrichment of almond milk with powder (target of 4% of protein)

* Best result obtain with ChickP G910

* No protein precipitation after heat treatment

* Firm gel with low syneresis after fermentation

* Need to add sucrose (2.5%) so that the strain can acidify enough (pH≈4,5)

* Sweet, slightly acidic, milky/yogurt taste

<figure><img src="image_77.png" /></figure>

<figure><img src="image_78.png" /></figure>

<figure><img src="image_79.png" /></figure>
# Task 2.3. Yogurt-type fermented probiotic gels

## Use of protein-enriched plant-based milk (almond/chickpea) supplied by Fraunhofer (3% of protein)

### Growth assessment

<figure><img src="image_80.png" /><figcaption>Population of *S. thermophilus* before and after fermentation depending on the type of milk analogues</figcaption></figure>

<figure><img src="image_81.png" /></figure>

Good acidification of milk analogues

Higher pH drop with chickpea (Higher carbohydrate concentration with the hydrolysis phase ? More free amino acid ?)

Growth of *S. thermophilus* (2-3 log)

Better growth with chickpea (Higher carbohydrate concentration with the hydrolysis phase ? More free amino acid ?)
# Task 2.3. Yogurt-type fermented probiotic gels

## Use of protein-enriched plant-based milk (almond/chickpea) supplied by Fraunhofer (3% of protein)

<table><thead><tr><th></th><th>100% Almond</th><th>75% Almond<br>25% Chickpea</th><th>50% Almond<br>50% Chickpea</th><th>25% Almond<br>75% Chickpea</th><th>100% Chickpea</th></tr></thead><tbody><tr><td>Appearance<br>of the gel</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Appearance<br>of the gel in<br>the spoon</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<figure><img src="image_82.png" /></figure>

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Not very firm gel and strong syneresis with 100% almond
Firm gel and low syneresis from 50% to 100% chickpea

<figure><img src="image_92.png" /></figure>
## Task 2.3. Yogurt-type fermented probiotic gels

### Use of protein-enriched plant-based milk (almond/chickpea) supplied by Fraunhofer (3% of protein)

#### "Sensory analysis" :

- 100% Almond : Milky, fresh, acid notes, appreciated

- 100% Chickpea : Notes of chickpea and fat, not appreciated

- Mixture almond/chickpea : Milky, fresh and acid notes, appreciated

#### Globally :

- Mixture (50/50) of the two milk analogues seems to be a good compromise : good smell and taste with good texture

- S. thermophilus was compared to an industrial yogurt starter (VegaClassic from Chr. Hansen) and was more appreciated (Better covers the legumes and roasted notes which were not appreciated)

<figure><img src="image_93.png" /></figure>
Thank you for your attention !

<figure><img src="image_94.png" /><figcaption>gwenael.jan@inrae.fr</figcaption></figure>

<figure><img src="image_95.png" /><figcaption>valerie.gagnaire@inrae.fr</figcaption></figure>

<figure><img src="image_96.png" /><figcaption>charles.silande@inrae.fr</figcaption></figure>

<figure><img src="image_97.png" /></figure>
## References

Illikoud N, Do Carmo FLR, Daniel N, Jan G, Gagnaire V. 2023. Development of innovative fermented products by exploiting the diversity of immunomodulatory properties and fermentative activity of lactic and propionic acid bacteria. *Food Research International* **166**: 112557.

<figure><img src="image_98.png" /></figure>