Comparison b/w 2 Case Studies on Soybean

In this presentation, comparison b/w 2 case studies i.e., Effect of Fermented soy beverage on Aged Female Mice Model and the other s Fluoride Exposure from Soybean Consumption: A Toxic Risk Assessment.

1. TITLE OF THE
PUBLICATION



JOURNAL OF
THE
PUBLICATION

PUBLICATION
DATE
EFFECT OF
FERMENTED SOY
BEVERAGE ON
AGED FEMALE
MICE MODEL



FOOD RESEARCH
INTERNATIONAL

www.elsevier.com/l
ocate/foodres.


5 APRIL 2023

FLUORIDE EXPOSURE
FROM SOYBEAN
CONSUMPTION: A
TOXIC RISK
ASSESSMENT


FOODS
MDPI

www.mdpi.com/journal/f
oods


17 JULY 2022
CASE
“A”
CASE
“B”

2. Effects of FSB enriched in bioactive compounds
due to the action of B.pseudocatenulatum INIA
P815, compared with SB in an aged vivo model.
Female aged mice.

Reproductive Similar to
(cyclic) menopause
(acyclic)

Investigations focus on:
Lipid Profile
Hepatic Gene Expression
Fecal Microbiota Composition
Inflammatory Profile
Fertility
Mice serum
Liver
Ovarian Fat


Analyzed the fluoride concentration in 30
samples in popular soy beverages (A,B,C-
brands respectively). \


Evaluate the toxicological risks derived from
its consumption considering the values of
ADI.
Risk assessment.


Possible statistical differences between the
soybean beverages

CASE
“A”
CASE
“B”
OBJECTIVE

3. WITHIN OUT OF
Female mice male mice
Matured,pre & post infant mice
Menopause
Vivo vitro
B.pseudocatenulatum ALL OTHER
INIA P815 CHEMICAL
Lipid profile COMPOUNDS
Hepatic gene expression
Fecal & oral microbiota
Composition
Inflammatory profile Colour, height
Fertility
Mice serum
Liver
Ovarian fat
WITHIN OUT OF
Soy Milk Dairy Milk
Toxicity benefits
Adverse effects Glass materials
Caused due to
Excess fluoride
Intake
Health effects
Ages 0-6 months
7-12 months
1-3 yrs
4-8 yrs
9-13 yrs
14-18 yrs
19-70 yrs
>70 yrs
Pregnancy lactation
14-50 yrs
Plastic apparatus

CASE
“A”
SCOPE
CASE
“B”

4. Fluoride handling in lab:

➢A 0.1 M stock fluoride solution was
created by dissolving sodium fluoride in
dried form.

➢Ultrapure distilled water was used from a
specific ultrafiltration system.

➢Glass materials were avoided due to
fluoride interaction with borosilicate
glass. Plastic containers, spatulas, and
volumetric flasks were used instead.

➢A 0.75 M orthophosphoric acid buffer
solution was prepared using
orthophosphoric acid dissolved in
distilled water, stored in a dark container








➢Bacterial Strain and Culture
Conditions:
Bacterial strain: B. pseudocatenulatum
INIA P815
Culture medium: MRS broth supplemented
with L-cysteine (MRS-cys)
Growth temperature: 37°C
Growth conditions: Anaerobic conditions
(10% H2, 10% CO2, and 80% N2)
Anaerobic workstation: Whitley DG250
Anaerobic workstation

➢FSB and SB Preparation and Analysis:
Soy beverage used: Commercial soy beverage
Vital (13% soy)
Stock inoculum preparation: B.
pseudocatenulatum grown in MRS-cys,
collected, and resuspended in SB


METHOD
CASE
“A”
CASE
“B”

5. Samples used:
➢The three most popular commercial
brands of soybean beverage that could be
purchased from the most common large
supermarkets were chosen.
➢A total of 30 samples of A-Brand,
B-Brand and C-Brand, from different
batches, were taken
Fluoride determination:
➢It was tested on 15 occasions over 3 days,
showing no significant differences in
results.
➢Accuracy was assessed by adding a
known fluoride amount to samples after
determining their concentration.
➢Mean recovery rate exceeded 99% with a
repeatability RSD of 0.60% and
reproducibility of 2.50%.











Fermentation process: SB inoculated with
stock inoculum, incubated anaerobically for
24 h at 37°C
Isoflavones and antioxidant activities
analyzed by HPLC-PAD and FRAP methods
Bacterial counts and pH measurements
performed before and after fermentation

➢Animal and Study Design:
96 female C57BL/6J mice, 10 months old
Divided into cyclic and acyclic groups
Housed with controlled temperature and
photoperiod
Basic diet low in phytoestrogens provided
Divided into control, SB, and FSB treatment
subgroups
Treatments administered for 36 days, with
measurements and samples taken periodically
METHOD
CASE
“A”

CASE
“B”

6. Method quality control and validity:
➢This study evaluated method precision
using repeatability and reproducibility
conditions. The method was tested across
three days, conducting 15 runs each day.
➢ Statistical analysis revealed consistent
results (p < 0.05) across testing days,
indicating precise performance.
➢To gauge method accuracy, a recovery
rate study added a known fluoride
amount to samples post-concentration
determination.
➢Measured vs. expected fluoride
concentrations were compared, revealing
insights into the method's accuracy and
reliability in quantifying sample
concentrations.












➢Fertility Assessment, Sacrifice, and
Organ Collection:
Superovulation induced with PMSG and hCG
Female mice crossed with males
Ovarian fat and ovaries weighed, oocytes and
zygotes counted
Liver and ovarian fat collected, weighed, and
frozen for analysis.

➢Isoflavones in Serum, Liver, and Ovarian
Fats:
Isoflavone extraction and analysis from serum,
liver, and ovarian fat samples
HPLC–ESI/MS used for analysis
Concentrations analyzed by ANOVA using
general linear model.

METHOD
CASE
“A”

CASE
“B”

7. Statistical analysis:
➢Statistical analysis was performed using
GraphPad Prism 9.0.2 software
(GraphPad Prism, San Diego, CA, USA)
➢First, we verified whether the values of
the analyzed samples followed a normal
distribution by applying the
Shapiro–Wilk test.
➢ However, since the data did not follow a
normal distribution, the non-parametric
statistical Kruskal–Wallis test was
applied.






➢Lipid Profile:
Total cholesterol and triglycerides measured
in mouse serum samples
Cholesterol and triglyceride concentrations
calculated from standard calibration curves.

➢Inflammatory Profile:
CRP, IL-6, and TNF-α levels measured in
mouse serum samples
Measured using ProcartaPlex Mouse Basic Kit
and Simplex Kits.

➢Microarray Analysis of Hepatic Gene
Expression:
Clariom S Arrays used for gene expression
analysis in the liver

METHOD
CASE
“A”

CASE
“B”

8. Fluoride intake risk assessment:
➢Calculates estimated fluoride ingestion
from a serving of soybean beverage,
typically a 200 mL glass.
➢The Estimated Daily Intake (EDI) is
obtained using the equation: EDI
(mg/day) = Mean consumption (L/day) ×
Fluoride concentration (mg/L).
➢Comparing with the Acceptable Daily
Intake (ADI), the equation is: ADI (%) =
[EDI/(ADI value)] × 100.
➢ A contribution over 10% is significant
for a single food, with over 50% possibly
indicating notable risk. Extreme risk
emerges when contribution nears or
surpasses 100% of ADI, especially with
prolonged consumption
Total RNA isolated and processed for
hybridization
DEGs identified and functional enrichment
analysis performed.

➢Faecal Microbiota:
Microbial composition analyzed using 16S
rRNA gene sequencing
Alpha and beta diversity metrics calculated
Taxonomic profiles studied from phylum to
species level
Statistical analyses performed using various
tests and packages.

METHOD
CASE
“A”

CASE
“B”

9. ➢Soybean beverage brands, notably
A-Brand, have high fluoride levels likely
due to sea salt.
➢Risky for kids due to fluoride. Ages >9
can have 3 servings of any brand, but
combined fluoride from other sources
could surpass limits.
➢>9 should stick to 1 serving, or pick
low-fluoride brand and limit to 3
servings.
➢Industry needs traceability and fluoride
control for raw materials. Control
crucial for soybean beverage quality and
safety
➢Fermentation Analysis (FSB and SB):
SB pH before fermentation: 6.94 ± 0.05
FSB pH after fermentation: 5.17 ± 0.20
SB isoflavones: Daidzin - 150.93 mg/L,
Genistin - 268.78 mg/L
FSB isoflavones: Daidzein - 209.58 mg/L,
Genistein - 197.13 mg/L
Antioxidant activity increase after
fermentation: 3.25 mg Trolox eq./mL
(fermented) vs. 2.67 mg Trolox eq./mL
(unfermented).

➢Effect on Body and Organs Weight:
No significant initial body weight differences
between mice groups





RESULT
CASE
“A”

CASE
“B”

10. SB and FSB treatment didn't affect body
weight
No significant differences in relative liver,
ovarian fat, and ovary weight between
treatments in cyclic or acyclic mice.
FSB treatment positively influenced retrieved
oocytes and zygotes in cyclic mice, not acyclic
mice

➢Isoflavone Presence:
Daidzein and genistein absent in control mice
serum
Detected in SB and FSB-treated mice serum,
no significant differences
Low levels of secondary compounds (DHD,
DHG, O-DMA, 6-OH-O-DMA) in SB and




➢The consumption of soybean beverages
and their fluoride intake was studied.
For ages 0-8, even one serving exceeded
safe fluoride limits, raising health
concerns.
➢In this age group, it's advised to avoid
soybean beverage consumption. For ages
9 and above, one serving doesn't pose
risks but contributes significantly to
fluoride intake.
➢Excessive consumption, alongside other
fluoride sources, could lead to health
issues. Moderate consumption is
recommended for all age groups due to
the risk of exceeding daily intake limits.

RESULT
CASE
“A”
CASE
“B”

11. FSB-treated mice serum
Daidzein and genistein present in liver and
ovarian fat of SB and FSB-treated mice at
low concentrations

➢Effect on Lipid Profile:
Total serum cholesterol showed no
significant differences after treatments
FSB group had lower serum triglycerides
than others
Significant difference to SB group only
in acyclic mice

➢Inflammatory Profile:
CRP levels:
No significant differences in cyclic mice
treated with SB and FSB vs. control



➢Table 3 shows C-Brand has the lowest
fluoride concentration (8.5 mg/L), while
B-Brand (11.3 mg/L) and A-Brand (15.5
mg/L) have higher concentrations.

➢Only C-Brand lacks sea salt, which can
raise fluoride levels due to impurities.
Despite sea salt typically having more
fluoride, a study found no significant
differences in fluoride content among
brands.

RESULT
CASE
“A”
CASE
“B”

12. In acyclic mice, FSB group had higher CRP
levels than SB group (P < 0.05)
IL-6 levels:
No differences observed between treatments
in both cyclic and acyclic groups
TNF-α levels:
FSB-treated mice had highest TNF-α levels in
both cyclic and acyclic groups
Significant increase (P < 0.05) in cyclic mice
treated with FSB compared to control

➢Hepatic Gene Expression Analysis:
Investigated effects of FSB and SB treatment
on hepatic gene expression using DNA
microarray analysis.
Paired comparison of groups led to
differentially expressed genes (DEGs) DEGs





➢All brands exceeded allowed
fluoride levels for water consumption
and recommendations.

➢Inconsistent levels within brands
highlight a lack of fluoride regulation
from ingredients to final product.

➢ Due to fluoride toxicity, authorities
should set legal limits for fluoride in
such products.







RESULT
CASE
“A”
CASE
“B”

13. were subjected to enrichment analysis using
DAVID, resulting in 27 significantly enriched
Biological Process (BP) Gene Ontology (GO)
terms Enriched GO terms included lipid
metabolic processes (fatty acid, steroid,
cholesterol, prostaglandin), showing varied
regulation in different comparisons.
Circadian processes also differentially
expressed in several comparisons.
Acyclic FSB vs SB mice comparison showed
highest number of enriched GO terms,
expressed in several comparisons.
Acyclic FSB vs SB mice comparison showed
highest number of enriched GO terms,
including xenobiotic compound metabolism
and stress response-related terms.

RESULT
CASE
“A”

14. RESULT
CASE
“A”
FSB vs control comparison had fewer
enriched GO terms for both cyclic and acyclic
mice.

➢Faecal Microbiota Analysis:
Alpha diversity indices calculated to display
differences in intestinal microbiota
composition of cyclic and acyclic mice in
each treatment group.
No significant differences found in richness
and diversity of microbial communities
within group and treatment Cyclic mice:
CNT > SB > FSB; SB showed least variation
between samples.
Acyclic mice: CNT showed highest diversity;
CNT and FSB had less variation between
samples than SB.

15. ➢Microbiota Analysis:
No significant differences observed in
Firmicutes/Bacteroidetes ratio between
treatments within cyclic and acyclic mouse
groups
Most abundant orders: Bacteroidales,
Lachnospirales, Clostridiales, Oscillospirales,
Erysipelotrichales, Saccharimonadales,
Lactobacillales
Changes within cyclic mice group:
SB: Decrease in Rhodospirillales, increase in
Pseudomonadales
Acyclic mice differences:
FSB: Increased Coriobacteriales and
Staphylococcales levels.
SB: Decreased Desulfovibrionales,
Acholeplasmatales, Lachnospirales;
RESULT
CASE
“A”

16. increased Erysipelotrichales
Family level differences
SB cyclic mice: Lower Bacteroidaceae,
increased Moraxellaceae.
FSB cyclic mice: Increased Streptococcaceae.
Acyclic mice differences observed in
Eggerthellaceae, Desulfovibrionaceae,
Acholeplasmataceae, Erysipelotrichaceae,
Gemellaceae, Lachnospiraceae families,
similar to corresponding orders.
SB acyclic mice: Increased Enterococcaceae,
[Eubacterium] coprostanoligenes group;
decreased Marinifilaceae.


RESULT
CASE
“A”