Microbial Inhibition Effect Report Yota-Guard™ CD — Cultured Dextrose
07/05/2026
Y
YOTABIO
Bio-Engineering Co., Ltd.
🔬 Laboratory ReportMIE-YGCD-2026-001
Microbial Inhibition Effect Report Yota-Guard™ CD — Cultured Dextrose
In Vitro Antimicrobial Activity Assessment Against Food-Relevant Molds, Bacteria, and Yeasts via Agar Diffusion, Minimum Inhibitory Concentration (MIC), and Time-Kill Kinetics
🔬 In Vitro StudyISO 20776-1Agar DiffusionMIC / MBCTime-KillDual-Acid SystemGLP Compliant
All cultures were sub-cultured twice prior to testing to ensure viability. Mold spore suspensions were prepared from 7-day-old PDA cultures and standardized using a hemocytometer. Bacterial inocula were prepared from overnight broth cultures and adjusted to McFarland 0.5 (≈1.5 × 10⁸ CFU/mL), then diluted to target levels.
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Test Methodology Summary
Method A — Agar Well Diffusion Ref: CLSI M44-A2 (mold) / M07-A11 (bacteria) Inoculated agar plates → 8 mm wells punched → 100 μL test solution added → incubated → zone of inhibition (ZOI) measured at 24/48/72 h. Each concentration tested in triplicate.
Method B — Minimum Inhibitory Concentration (MIC) Ref: ISO 20776-1 / CLSI M38 (mold) / M07 (bacteria) Broth microdilution in 96-well plates. Serial 2-fold dilutions of test article (0.0125–5.0 % w/v). Inoculated → incubated → MIC = lowest conc. with no visible growth. MBC/MFC = sub-cultured onto fresh agar.
Method C — Time-Kill Kinetics Ref: CLSI M26-A Selected organisms exposed to 1× and 2× MIC in liquid media. Viable counts (CFU/mL) taken at 0, 2, 4, 8, 12, 24, 48 h. ≥3-log₁₀ reduction = bactericidal/fungicidal. All tests at pH 5.0 and 5.5.
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Results — Agar Well Diffusion (Zone of Inhibition, mm)
Test solutions at 2.0 % (w/v) active concentration, pH adjusted to 5.0. Zones measured as diameter (mm) including the 8 mm well. Mean of 3 replicates ± SD.
Organism
Yota-Guard CD 2.0 %
Ca-Propionate 2.0 %
K-Sorbate 2.0 %
Na-Benzoate 2.0 %
Neg. Control (Water)
Interpretation
Aspergillus niger
28.3 ± 1.2
24.1 ± 0.8
19.7 ± 1.0
12.5 ± 0.6
0
Superior to all controls
Penicillium chrysogenum
30.7 ± 1.5
26.2 ± 1.1
22.4 ± 0.9
13.8 ± 0.7
0
Superior to all controls
Penicillium roqueforti
27.5 ± 0.9
23.8 ± 1.0
20.1 ± 1.2
11.2 ± 0.5
0
Superior to all controls
Rhizopus stolonifer
32.1 ± 1.8
27.5 ± 1.3
18.3 ± 1.1
10.6 ± 0.8
0
Markedly superior
Cladosporium spp.
26.4 ± 1.0
22.7 ± 0.9
21.5 ± 1.3
14.1 ± 0.6
0
Superior to all controls
Bacillus subtilis (rope)
25.8 ± 1.4
18.2 ± 1.0
10.3 ± 0.7
8.5 ± 0.4
0
Significantly superior (+42 %)
Bacillus licheniformis
23.6 ± 1.1
16.9 ± 0.8
9.8 ± 0.6
8.2 ± 0.5
0
Significantly superior (+40 %)
Listeria monocytogenes
22.1 ± 0.7
14.5 ± 0.6
16.7 ± 0.9
18.3 ± 0.8
0
+52 % vs Ca-propionate
E. coli O157:H7
18.9 ± 0.8
12.1 ± 0.5
14.2 ± 0.7
19.5 ± 1.0
0
Moderate — enhanced vs CaProp
S. cerevisiae (baker's yeast)
0 ± 0
0 ± 0
21.8 ± 1.2
15.3 ± 0.9
0
✅ NO yeast inhibition
Key Finding — Agar Diffusion: Yota-Guard CD at 2.0 % produced inhibition zones 15–52 % larger than equivalent calcium propionate across all molds and bacteria. The most dramatic advantage was against B. subtilis (rope) and Listeria, attributable to the 12.7 % acetic acid content acting synergistically with propionic acid. Critically, zero inhibition of S. cerevisiae confirmed complete yeast safety — unlike potassium sorbate which inhibited yeast (ZOI = 21.8 mm).
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Results — MIC & MFC/MBC Determination
Broth microdilution at pH 5.0 (bread-relevant). Values in % (w/v). MIC = no visible growth after 24 h (bacteria) or 72 h (molds). MFC/MBC confirmed by sub-culture (≥99.9 % kill).
Organism
Yota-Guard™ CD
Ca-Propionate
K-Sorbate
Na-Benzoate
YG-CD vs CaProp
MIC %
MFC/MBC %
MIC %
MFC/MBC %
MIC %
MFC/MBC %
MIC %
MFC/MBC %
Aspergillus niger
0.25
0.50
0.15
0.30
0.10
0.25
0.50
1.00
~1.7× (weight) / equiv. active
P. chrysogenum
0.20
0.40
0.12
0.25
0.08
0.20
0.45
0.80
~1.7× / equivalent active
P. roqueforti
0.25
0.50
0.15
0.35
0.10
0.22
0.55
1.00
~1.7× / equivalent active
R. stolonifer
0.15
0.30
0.10
0.20
0.12
0.30
0.60
1.20
1.5× / superior
Cladosporium spp.
0.30
0.60
0.18
0.40
0.10
0.25
0.40
0.90
~1.7× / equivalent active
B. subtilis (rope)
0.20
0.40
0.20
0.50
0.80
>2.0
0.60
1.20
Equal MIC / superior MBC
B. licheniformis
0.25
0.50
0.22
0.55
1.00
>2.0
0.70
1.50
~Equal MIC / superior MBC
L. monocytogenes
0.15
0.30
0.25
0.60
0.20
0.50
0.15
0.35
40 % lower MIC
E. coli O157:H7
0.40
0.80
0.50
1.20
0.30
0.70
0.10
0.25
20 % lower MIC
S. cerevisiae
> 5.0
> 5.0
> 5.0
> 5.0
0.05
0.15
0.25
0.50
✅ NO inhibition up to 5 %
✅ MIC Data — Key Conclusions: 1) On a weight basis, YG-CD MIC values are ~1.5–1.7× those of pure CaProp — fully consistent with the 2:1 dosage ratio (YG-CD is ~42% propionic acid vs 100% for reagent CaProp). 2) Against Bacillus rope organisms, YG-CD shows equal or lower MIC and significantly lower MBC — the acetic acid (12.7%) provides bactericidal synergy not available from propionic acid alone. 3) Against Listeria, YG-CD MIC was 40% lower than CaProp — a major advantage for RTE applications. 4) Baker's yeast showed no inhibition at concentrations up to 5.0 % — far exceeding any practical bakery dosage.
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pH-Dependent MIC Variation — Yota-Guard™ CD
MIC values (% w/v) for Yota-Guard CD tested across pH 4.5 – 6.0. Lower pH = more undissociated acid = lower MIC. Efficacy governed by application matrix pH.
Organism
MIC at pH 4.5
MIC at pH 5.0
MIC at pH 5.5
MIC at pH 6.0
Fold Change (pH 4.5 → 6.0)
Practical Implication
Aspergillus niger
0.10
0.25
0.50
1.20
12×
Lowering pH is critical
P. chrysogenum
0.08
0.20
0.45
1.00
12.5×
Optimum at bread pH ≤ 5.0
R. stolonifer
0.06
0.15
0.35
0.90
15×
Most pH-sensitive species
Cladosporium spp.
0.12
0.30
0.55
1.30
10.8×
Standard pH dependency
B. subtilis (rope)
0.08
0.20
0.50
1.50
18.8×
Acetic acid enhances anti-rope at all pH
L. monocytogenes
0.06
0.15
0.40
1.00
16.7×
Dual-acid synergy critical for RTE
E. coli O157:H7
0.15
0.40
0.90
2.50
16.7×
Requires pH ≤ 5.0 for optimal effect
S. cerevisiae
> 5.0
> 5.0
> 5.0
> 5.0
N/A
✅ Yeast-safe at ALL pH levels
⚠️ Critical pH Insight: MIC increases 10–19× from pH 4.5 to pH 6.0 across all organisms. For products with pH > 5.5, formulators should either (a) increase dosage by 50–100 %, (b) combine with acidulant to lower matrix pH, or (c) apply as post-bake surface spray. Baker's yeast remains unaffected even at 5.0 % regardless of pH.
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Results — Time-Kill Kinetics (Selected Organisms)
Log₁₀ CFU/mL at specified time points. Test concentration = 2× MIC (at pH 5.0). A ≥ 3-log₁₀ reduction from T₀ = fungicidal/bactericidal activity.
Panel A — Mold Time-Kill (Spore Germination Inhibition)
Time (h)
A. niger YG-CD
A. niger CaProp
P. chrysogenum YG-CD
P. chrysogenum CaProp
R. stolonifer YG-CD
R. stolonifer CaProp
T₀
6.00
6.00
6.00
6.00
6.00
6.00
4 h
5.95
5.98
5.90
5.96
5.82
5.95
8 h
5.70
5.88
5.55
5.80
5.40
5.78
12 h
5.20
5.65
4.85
5.50
4.60
5.45
24 h
4.10
5.10
3.80
4.80
3.20
4.60
48 h
2.50
4.20
2.10
3.90
1.50
3.50
72 h
< 1.0
3.40
< 1.0
3.10
< 1.0
2.80
Log₁₀ Reduction
> 5.0
2.6
> 5.0
2.9
> 5.0
3.2
Classification
Fungicidal
Fungistatic
Fungicidal
Fungistatic
Fungicidal
Fungicidal
Panel B — Bacterial Time-Kill
Time (h)
B. subtilis YG-CD
B. subtilis CaProp
Listeria YG-CD
Listeria CaProp
E. coli YG-CD
E. coli CaProp
T₀
6.00
6.00
6.00
6.00
6.00
6.00
2 h
5.60
5.85
5.40
5.80
5.70
5.90
4 h
4.80
5.50
4.50
5.55
5.20
5.70
8 h
3.50
4.80
3.20
5.00
4.30
5.30
12 h
2.20
4.20
1.80
4.50
3.40
4.80
24 h
< 1.0
3.50
< 1.0
3.80
2.10
4.20
Log₁₀ Reduction
> 5.0
2.5
> 5.0
2.2
3.9
1.8
Classification
Bactericidal
Bacteriostatic
Bactericidal
Bacteriostatic
Bactericidal
Bacteriostatic
Time-Kill: A. niger — YG-CD vs Ca-Propionate (pH 5.0, 2×MIC)
YG-CD achieved fungicidal kill (>5 log₁₀) by 72 h; CaProp remained fungistatic (2.6 log₁₀).
✅ Time-Kill Conclusions: At 2× MIC, Yota-Guard CD achieved fungicidal activity (> 5-log₁₀ reduction) against all three key bread molds within 72 h — whereas calcium propionate was merely fungistatic. Against B. subtilis (rope) and Listeria, YG-CD was bactericidal within 24 h, while CaProp was only bacteriostatic. This difference is attributable to the dual-acid mechanism: propionic acid provides primary antifungal activity while acetic acid (12.7 %) delivers synergistic membrane disruption.
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Dual-Acid Synergy Analysis — FICI Checkerboard
Fractional Inhibitory Concentration Index (FICI) calculated using checkerboard assay at pH 5.0.
🔬 Synergy Conclusion: True synergy (FICI ≤ 0.50) was confirmed specifically against Bacillus rope organisms and Listeria monocytogenes. Against molds, the interaction is additive/partially synergistic (FICI 0.67–0.72). Yota-Guard CD's naturally co-produced acetic acid is not merely a bystander — it actively reduces the amount of propionic acid needed for bacterial inhibition.
Fermentation impact study: S. cerevisiae in standard lean dough formula at 30 °C, measuring CO₂ production (mL) over 120 min.
Additive
Dose (%FW)
CO₂ @ 30 min
CO₂ @ 60 min
CO₂ @ 90 min
CO₂ @ 120 min
% of Control
Impact
Control (no additive)
—
85
210
380
520
100.0 %
—
Yota-Guard CD
0.40
84
208
378
516
99.2 %
None
Yota-Guard CD
0.60
83
205
373
510
98.1 %
None
Yota-Guard CD
0.80
82
202
368
502
96.5 %
Negligible
Yota-Guard CD
1.00
80
198
360
492
94.6 %
Negligible
Yota-Guard CD
1.50
77
190
345
471
90.6 %
Minor (−9.4 %)
Ca-Propionate
0.30
82
203
370
505
97.1 %
Negligible
K-Sorbate
0.10
50
115
190
260
50.0 %
Severe (−50 %)
K-Sorbate
0.20
22
45
68
90
17.3 %
Critical (−83 %)
✅ Yeast Safety Confirmed: At the maximum recommended bakery dosage (1.0 % FW), Yota-Guard CD reduced CO₂ production by only 5.4 % — no practical impact on proofing time or dough volume. In contrast, potassium sorbate at just 0.10 % caused a 50 % reduction — making it incompatible with yeast-leavened products.
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Statistical Summary & Quality Parameters
Study Quality Metrics
Total Tests Conducted
486 (incl. replicates)
Organisms Tested
10 strains (5 molds, 4 bacteria, 1 yeast)
Methods Employed
3 (Agar Diffusion, MIC/MFC/MBC, Time-Kill)
pH Conditions
4 levels (4.5, 5.0, 5.5, 6.0)
Replicates per Test
3 (minimum); 5 for key MIC determinations
Statistical Analysis
ANOVA with Tukey HSD post-hoc (α = 0.05)
All Results Significant?
YES (p < 0.05)
Coefficient of Variation
≤ 8.5 % (all assays)
Overall Efficacy Summary
Anti-Mold (5 species)
★★★★★ EXCELLENT
Anti-Rope (Bacillus)
★★★★★ EXCELLENT (Synergistic)
Anti-Listeria
★★★★★ EXCELLENT (Synergistic)
Anti-E. coli
★★★★☆ VERY GOOD
Yeast Safety
★★★★★ FULLY COMPATIBLE
vs. Ca-Propionate
✅ Superior (at 2:1 ratio)
vs. K-Sorbate
✅ Superior (no yeast kill)
vs. Na-Benzoate
✅ Superior (broader spectrum)
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Conclusions
#
Conclusion
Significance
1
Yota-Guard CD demonstrates broad-spectrum antimicrobial activity against all five tested mold species, two Bacillus rope organisms, Listeria monocytogenes, and E. coli O157:H7.
Confirmed
2
On an equal active-acid basis, Yota-Guard CD delivers inhibition equivalent to or exceeding pure calcium propionate. The 2:1 weight replacement ratio is validated.
Validated
3
The naturally co-produced acetic acid (12.7 %) provides true synergy (FICI ≤ 0.50) with propionic acid against Bacillus rope and Listeria.
Major Advantage
4
At 2× MIC, Yota-Guard CD is fungicidal and bactericidal (≥ 5-log₁₀ kill), whereas CaProp is merely fungistatic/bacteriostatic (2–3 log₁₀).
Superior Kill
5
Zero inhibition of baker's yeast at concentrations up to 5.0 %. Fermentation output at max recommended dose (1.0 %) was 94.6 % of control.
