Sourdough Fermented Breads are More Digestible [626857]

nutrients
Article
Sourdough Fermented Breads are More Digestible
than Those Started with Baker’s Yeast Alone: An In
Vivo Challenge Dissecting Distinct
Gastrointestinal Responses
Carlo Giuseppe Rizzello1,*,y
, Piero Portincasa2,*,y
, Marco Montemurro1
,
Domenica Maria Di Palo1,2, Michele Pio Lorusso1, Maria De Angelis1
, Leonilde Bonfrate2,
Bernard Genot3and Marco Gobbetti4
1Department of Soil, Plant and Food Science, University of Bari Aldo Moro, 70126 Bari, Italy;
[anonimizat] (M.M.); [anonimizat] (D.M.D.P .); [anonimizat] (M.P .L.);
[anonimizat] (M.D.A.)
2Clinica Medica “A. Murri”, Department of Biomedical Sciences & Human Oncology, University of Bari
Medical School, 70124 Bari, Italy; [anonimizat]
3Puratos NV , 1702 Groot-Bijgaarden, Belgium; [anonimizat]
4Faculty of Science and Technology, Free University of Bozen-Bolzano, 39100 Bolzano, Italy;
[anonimizat]
*Correspondence: [anonimizat] (C.G.R.); [anonimizat] (P .P .);
Tel.:+39-080542945 (C.G.R.); +39-0805478227 (P .P .)
yThe two authors contributed equally.
Received: 9 September 2019; Accepted: 26 November 2019; Published: 4 December 2019
/gid00030/gid00035/gid00032/gid00030/gid00038/gid00001/gid00033/gid00042/gid00045 /gid00001
/gid00048/gid00043/gid00031/gid00028/gid00047/gid00032/gid00046
Abstract: As a staple food, bread digestibility deserves a marked nutritional interest. Combining
wide-spectrum characterization of breads, in vitro nutritional indices, and in vivo postprandial
markers of gastrointestinal function, we aimed at comparing the digestibility of sourdough and
baker’s yeast breads. Microbiological and biochemical data showed the representativeness of the
baker ´s yeast bread (BYB) and the two sourdough breads (SB and t-SB, mainly di ering for the
time of fermentation) manufactured at semi-industrial level. All in vitro nutritional indices had the
highest scores for sourdough breads. Thirty-six healthy volunteers underwent an in vivo challenge
in response to bread ingestion, while monitoring gallbladder, stomach, and oro-cecal motility. SB,
made with moderate sourdough acidification, stimulated more appetite and induced lower satiety.
t-SB, having the most intense acidic taste, induced the highest fullness perception in the shortest time.
Gallbladder response did not di er among breads, while gastric emptying was faster with sourdough
breads. Oro-cecal transit was prolonged for BYB and faster for sourdough breads, especially when
made with traditional and long-time fermentation (t-SB), whose transit lasted ca. 20 min less than BYB.
Dierences in carbohydrate digestibility and absorption determined di erent post-prandial glycaemia
responses. Sourdough breads had the lowest values. After ingesting sourdough breads, which had a
concentration of total free amino acids markedly higher than that of BYB, the levels in blood plasma
were maintained at constantly high levels for extended time.
Keywords: gallbladder emptying; hydrogen breath test; stomach emptying; orocecal transit time;
test meal; ultrasonography
1. Introduction
Bread has been and is one of the worldwide-consumed staple foods [ 1]. A bread recipe comprises
cereal flour (e.g., wheat, rye), even if the use of pseudo-cereals and /or legumes is increasing, tap
Nutrients 2019 ,11, 2954; doi:10.3390 /nu11122954 www.mdpi.com /journal /nutrients

Nutrients 2019 ,11, 2954 2 of 21
water, eventually salt and other minor ingredients, and a leavening agent, responsible for gas release
and consequently dough expansion. The choice of the leavening agent is crucial for a number of
technological factors (e.g., costs and time of processing) and, primarily, because of the repercussions
on the bread sensory, texture, nutritional and shelf life features. Commonly, three types of leavening
agents may be used: chemicals, baker’s yeast (e.g., commercial preparations of Saccharomyces cerevisiae
cells), and sourdough. While the production and commercialization of the first two types takes place at
industrial levels, the sourdough is an old example of natural starter, the artisanal use of which complies
with regional traditions and cultural heritages. Sourdough is a mixture of flour, water and other
ingredients (e.g., salt), which is fermented by naturally occurring lactic acid bacteria and yeasts that
propagate during back slopping, a traditional procedure in which the sourdough from the previous
fermentation cycle is used as the starter to ferment a new mixture of flour and water [2].
Lactic acid bacteria and yeasts come from flour and house microbiota, but their shaping and
assembly into a mature biota during fermentation depend on many drivers, such as the chemical and
enzyme composition of the flour, and the temperature, redox potential, water content and duration
of the process [ 3]. Lactic acid bacteria dominate the mature sourdough, reaching a cell density >108
cfu/g [4]. The number of yeasts is one /two logarithmic cycles lower [ 5]. In the second half of the
last century, fast leavening processes by chemicals and /or baker’s yeast almost replaced the use of
sourdough. Using these leavening agents, the main polymeric cereal components (e.g., proteins, starch)
undergo very mild or absent hydrolysis during processing. Such biochemical events deeply a ect
the bioavailability of nutrients and lead to the release of bioactive and aroma compounds [ 6–8]. A
renewed scientific interest in sourdough has developed during the last decades. The Scopus database
(November 2019) reports approximately 1400 published items on sourdough biotechnology during the
last fifteen years. Compared to the other leavening agents, the sourdough has the capability to positively
influence the bread sensory, texture, nutritional and shelf-life features [ 7,8]. The e ects of sourdough
fermentation are related to organic acids synthesis, the activation of the endogenous enzymes of the
flour as well as the synthesis of microbial secondary metabolites. Among the main advantages related
to sourdough use, the increase of the in vitro protein digestibility and amount of soluble fibre, and the
decrease of the glycaemic index, phytate content, trypsin inhibitors, and other anti-nutritional factors,
have been described [ 9]. These scientific evidences promoted its technology transfer and favoured an
increasing global manufacture of sourdough breads, which reflect the ancient tradition and satisfy the
consumer expectations for natural, high nutritious and sustainable foods [ 10,11]. The current European
manufacture of sourdough breads covers 30–50% of the global market and strengthens this renewed
interest by industries, artisans and consumers [12].
Traditional, empirical and in vitro scientific results all agree that sourdough and, more in general,
the long-time fermentation processes are associated with an improved bread digestibility. Nevertheless,
to define univocally the concept of bread digestibility from a nutritional perspective seems controversial.
Indeed, bread digestibility relies on factors of di erent nature: the perception of appetite, satiety
and gastrointestinal symptoms after ingestion [ 13–15], and the bioavailability of proteins and starch.
A few in vivo clinical studies have already demonstrated the e ect of sourdough fermentation on
starch digestibility. Compared to breads leavened with chemicals or baker’s yeast, the digestible starch
fraction of sourdough breads significantly decreased [ 16,17]. On the contrary, only in vitro data have
accumulated for protein digestibility [ 18]. An in vivo challenge focusing the overall sourdough bread
digestibility, which strengthens or not the empirical and the in vitro scientific evidences, is still missing.
First, breads obtained by baker’s yeast or sourdough fermentation were subjected to an extensive
in vitro biochemical characterization. Then, an in vivo clinical study based on complementary
techniques of investigation to assess the e ect of fermentation on bread digestibility was carried out.
The study comprised gastrointestinal motility in response to di erent breads, and the comparison with
a reference test meal.

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2. Materials and Methods
2.1. Microorganisms and Flour
Lactobacillus plantarum CR1, Lactobacillus rossiae CR5 and Saccharomyces cerevisiae E10, belonging to
the Culture Collection of the Department of Soil, Plant and Food Sciences (University of Bari, Italy),
were inoculated into the wheat flour dough used for preparing the sourdough. Lactic acid bacteria
were propagated for 24 h at 30C on modified MRS broth (Oxoid, Basingstoke, Hampshire, United
Kingdom), with the addition of fresh yeast extract (5%, v/v) and 28 mM maltose, at the final pH of
5.6 (mMRS). The propagation of S. cerevisiae E10 was at 30C for 48 h on Sabouraud dextrose broth
(Oxoid). When used for fermentation, cells of lactic acid bacteria and yeasts were cultivated until
reaching the late exponential phase of growth (ca. 12 and 24 h, respectively). Cell suspensions for the
inoculum were prepared according to Rizzello et al. [19].
The gross chemical characteristics of the wheat ( Triticum aestivum cv. Appulo) flour used for
making sourdoughs and breads were as follows: moisture, 13.2%; protein, 9.5%; fat, 1.2%; ash, 0.4%;
and total carbohydrates, 73.5%.
2.2. Sourdough Preparation
A type I sourdough was made and propagated through a traditional protocol [ 20,21], starting
from a fermented dough inoculated with Lb. plantarum CR1, Lb. rossiae CR5 and S. cerevisiae E10.
Wheat flour was mixed with tap water, containing cell suspensions, at 60 rpm for 5 min with a IM
5-8 high-speed mixer (Mecnosud, Flumeri, Italy), and the dough (Dough Yield (DY) =(dough /flour
weight)100=of 160) was incubated at 30C for 16 h. The inoculum corresponded to ca. 5 107and
ca. 5106cfu/g for lactic acid bacteria and yeasts, respectively. Further the first fermentation, four
back slopping steps (refreshments) were carried out, mixing 20% of the previously fermented dough
with flour and water (DY of 160), and incubating for 8 h at 30C. After each fermentation, doughs were
stored at 4C until the next refreshment. The value of pH of the doughs was determined by a pHmeter
(Model 507, Crison, Milan, Italy) with a food penetration probe. Total titratable acidity (TTA) was
determined after homogenization of 10 g of dough with 90 mL of distilled water and expressed as the
amount (mL) of 0.1 M NaOH required to neutralize the solution, using phenolphthalein as indicator
(ocial American Association for Clinical Chemistry: AACC method 02-31.01). The rate of volume
increase of doughs was determined as described by Minervini et al. [ 22]. After four refreshments, the
acidification rate and volume increase of the dough were stable, and the mature sourdough (S) was
used for preparing the sourdoughs to be used in bread making. In detail, S 4and S 24were prepared
after refreshing (mixing 20% of S with flour and water, DY of 160) at 30C for 4 and 24 h, respectively.
2.3. Sourdoughs Characterization
For microbiological analyses, 10 g of each sourdough were homogenized with 90 mL of sterile
peptone water (1% ( w/v) of peptone and 0.9% ( w/v) of NaCl) solution. Presumptive lactic acid bacteria
were enumerated on MRS (Oxoid) supplemented with cycloheximide (0.1 g liter). Plates were incubated
at 30C for 48 h, under anaerobiosis (AnaeroGen and AnaeroJar, Oxoid). Yeasts were enumerated on
Sabouraud dextrose agar (SDA) (Oxoid) medium supplemented with chloramphenicol (0.1 g /L) at
30C for 48 h.
Water /salt-soluble extracts (WSE) of sourdoughs were prepared according to Weiss et al. [ 23] and
used to analyse free amino acids (FAA) and organic acids. FAA were analysed by a Biochrom 30 series
Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England) with a Na-cation-exchange
column (20 by 0.46 cm internal diameter), as described by Rizzello et al. [ 19]. Organic acids were
determined by High Performance Liquid Chromatography (HPLC), using an ÄKTA Purifier system
(GE Healthcare, Buckinghmshire, UK) equipped with an Aminex HPX-87H column (ion exclusion,
Biorad, Richmond, CA, USA), and an UV detector operating at 210 nm. Elution was at 60C, with a
flow rate of 0.6 mL /min, using H 2SO410 mM as mobile phase [ 24]. The Fermentation Quotient (FQ)

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was determined as the molar ratio between lactic and acetic acids. Fermentations were carried out in
triplicate and each one was analysed in duplicate.
2.4. Bread Making
Industrial breads were manufactured at the pilot plant of ValleFiorita s.r.l. (Ostuni, Italy). Three
types of bread (ca. 500 g each one, Supplementary Figure S1) were manufactured. BYB was made
mixing (60gfor 5 min, IM 5-8 high-speed mixer, Mecnosud, Flumeri, Italy) wheat flour (62.5% w/w),
water (37.5% w/w) and 1.5% ( w/w) of baker’s yeast. The fermentation lasted 2 h at 30C. SB was
manufactured according to a two-stage protocol, which is routinely used in artisanal and industrial
bakeries [ 25]. The formula consists of 20% ( w/w) sourdough S 4(fermented for 4 h at 30C, step I),
which was then mixed (see above) with flour (50% w/w), water (30% w/w) and 1.5% baker’s yeast,
and further incubated for 1.5 h at 30C (step II). The third type of bread was the most traditional (t-SB).
It was manufactured according to the above two-stage protocol but without baker’s yeast. The formula
consists of 20% ( w/w) sourdough S 24(fermented for 24 h at 30C, step I), which was then mixed (see
above) with flour (50% w/w) and water (30% w/w), and further incubated for 4 h at 30C (step II). The
most common percentage (20%, w/w) used at industrial /artisanal levels was chosen to inoculate S 4
and S 24[25,26]. All breads were baked at 220C for 30 min (Combo 3, Zucchelli, Verona, Italy). Bread
making was carried out in triplicate and each bread was analysed twice.
2.5. Biochemical, Textural and Nnutritional Characterization of Breads
The values of pH, TTA, organic acids and FAA were determined on fermented doughs before
baking, as described elsewhere. Gluten content was determined on doughs before baking by using
Glutomatic 2200 following AACC Method 38-12. Protein (total nitrogen 5.7), lipids and ash were
determined according to the AACC o cial methods 46-11A, 30-10.01, and 08-01, respectively while total
carbohydrates were calculated as the di erence (100(proteins +lipids +ash)). The determination of
dietary fibre was carried out by Association of O cial Analytical Chemists (AOAC) approved methods
991.43. Energy value was determined according to FAO guidelines [ 27]. Breads proximal composition
and energy value are reported in Supplementary Table S1.
Instrumental Texture Profile Analysis (TPA) was carried out with a TVT-300XP Texture Analyser
(TexVol Instruments, Viken, Sweden), equipped with a cylinder probe P-Cy25S. For this analysis, boule
shaped loaves (300 g) were baked, packed in polypropylene micro perforated bags and stored for
24 h at room temperature. Crust was not removed. The selected settings were as follows: test speed
1 mm /s, 30% deformation of the sample and one compression cycle. TPA [ 28] was carried out using
Texture Analyzer TVT-XP 3.8.0.5 software (TexVol Perten Instruments, Hägersten, Sweden). Bread
height, width, depth, area and specific volume were measured through the BVM-test system (TexVol
Instruments). The following textural parameters were obtained: hardness (maximum peak force);
fracturability (the first significant peak force during the probe compression of the bread); and resilience
(ratio of the first decompression area to the first compression area). The chromaticity co-ordinates of
the bread crust (Minolta CR-10 camera) were also reported in the form of a colour di erence, dE * ab,
as follows:
dEab=q
(dL)2+(da)2+(db)2, (1)
where dL,da, and dbare the di erences for L,a, and bvalues between sample and reference (a white
ceramic plate having L=93.4, a=0.39, and b=3.99). The bread crumb features were assessed after
24 h of storage using the image analysis technology with the UTHSCSA ImageTool, as previously
described by Rizzello et al. [28].
The In Vitro Protein Digestibility (IVPD) of flours, sourdoughs and breads was determined by
the method proposed by Akeson and Stahmann [ 29], with some modifications [ 18]. Samples were
subjected to a sequential enzyme treatment mimicking the in vivo digestion in the gastro intestinal
tract and IVPD was expressed as the percentage of the total protein which was solubilized after enzyme

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hydrolysis. The supernatant, which contained the digested proteins, was freeze-dried and used for
further analyses. The modified method AOAC 982.30a was used to determine the total amino acid
profile [ 30]. The digested protein fraction, which derived from 1 g of sample, was added of 5.7 M
HCl (1 mL /10 mg of proteins), under nitrogen stream, and incubated at 110C for 24 h. Hydrolysis
was carried out under anaerobic conditions to prevent the oxidative degradation of amino acids.
After freeze-drying, the hydrolysate was re-suspended (20 mg /mL) in sodium citrate bu er, pH 2.2,
and filtered through a Millex-HA 0.22 m pore size filter (Millipore Co.). Amino acids were analysed
by a Biochrom 30 series Amino Acid Analyzer as described above. Because this procedure of hydrolysis
does not allow the determination of tryptophan, this amino acid was estimated by the method of
Pintér-Szak ács and Moln án-Perl [ 31]. One gram of sample was suspended into 10 mL of 75 mM NaOH
and shaken for 30 min at room temperature. After centrifugation (10,000 rpm for 10 min), 0.5 mL of the
supernatant were mixed with 5 mL of ninhydrin reagent (1 g of ninhydrin in 100 mL of a solution HCl
37% and formic acid 96%, ratio 2:3) and incubated for 2 h at 37C. The reaction mixture was cooled at
room temperature and made up to 10 mL with the addition of diethyl ether. The absorbance at 380
nm was measured. A standard tryptophan curve was prepared using a tryptophan (Sigma Chemicals
Co.) solution in the range 0–100 g/mL. Chemical Score (CS) estimates the amount of protein required
to provide the minimal EAA pattern for adults, which was recently re-defined by FAO (Food and
Agriculture Organization) in 2007 [ 32]. CS was calculated using the equation of Block and Mitchel [ 33],
which compares the ratio of the individual essential amino acid (EAA) in the bread protein to that
of the corresponding amino acid in the reference. The sequence of limiting essential amino acids
corresponds to the list of EAA, having the lowest chemical score. The protein score indicates the
chemical score of the most limiting EAA that is present in the test protein. Essential Amino Acids
Index (EAAI) estimates the quality of the test protein, using its EAA content as the criterion. EAAI
was calculated according to the procedure of Oser [ 34]. It considers the ratio between EAA of the test
protein and EAA of the reference protein, according to the following equation:
EAAI =ns
(EAA 1100)(EAA 2100)(: : :)(EAA n100)(sample )
(EAA 1100)(EAA 2100)(: : :)(EAA n100)(re f erence ). (2)
The biological value (BV) indicates the utilizable fraction of the test protein. BV was calculated
using the equation of Oser [ 34]: BV =((1.09EAAI)11.70). The Protein E ciency Ratio (PER)
estimates the protein nutritional quality based on the amino acid profile after hydrolysis. PER was
determined using the model developed by Ihekoronye [ 35]: PER =0.468 +(0.454(Leucine))
(0.105(Tyrosine)). The NI normalizes the qualitative and quantitative variations of the test protein
compared to its nutritional status. NI was calculated using the equation of Crisan and Sands [ 36],
which considers all the factors with an equal importance: NI =(EAAIProtein (%) /100).
2.6. Volunteers Enrolment and Test Meal Administration
The sample size was calculated assuming a 10% di erence in response quantitative perception
and motility. We estimated that 30 patients would be required for the study to have 90% power and
anerror of 5% (Sigmaplot v. 14.0, Systat Software, San Jose, CA, USA). Thus, 36 healthy subjects
(20–31 years old; sex 18 male : 18 female; Body Mass Index, e.g., <25 kg /m2) were recruited at a
tertiary referral center (Clinica Medica “A. Murri”, Department of Biomedical Science and Human
Oncology, University of Bari). All subjects gave their informed consent and, at entry, underwent a
full clinical anamnesis to exclude clinically evident diseases. Exclusion criteria were diagnosis of
organic diseases, and therapies potentially influencing sensory perception or gastrointestinal motility.
Volunteers did not show gastrointestinal symptoms in the three months before the trial and none
had a previous history of gastrointestinal disease or surgery. The study was no-profit and approved
by the Ethics Review Board of the University Hospital Policlinico in Bari (code: natural bread 2017;
n. 122 /18). All experiments were performed according to European Community and local Ethics

Nutrients 2019 ,11, 2954 6 of 21
Review Board guidelines. All experiments started at 8.00 a.m. after an overnight fast of at least 12 h.
Antibiotics, probiotics or other drugs known to a ect gastrointestinal motility or intestinal microbiota
were prohibited from 10 days before the trial. To avoid prolonged intestinal H 2-production due to
the secondary presence of non-absorbable or slowly fermentable food, a diet, including meat, fish,
eggs and olive oil, and water as drink [ 37] was prescribed starting from lunchtime, the day before
the test. Any other fermentable food or sweet beverage was prohibited before and during the test.
The test meal comprised one slice of bread (80 g) (equivalent to 220 kcal, with 8% protein, 0.5% fat,
63.5% carbohydrates, of which 56% starch), which was administered together with a glass of 160 mL
of water plus 10 g of lactulose powder (Duphalac Dry®, Solvay Pharma, Turin, Italy). The lactulose
is a non-absorbable substrate but fermentable to hydrogen (H 2) by the resident oro-cecal microbiota.
This fermentation is a crude estimate of the Oro-Cecal Transit Time (OCTT) [ 37]. The solution, at
room temperature, was administered over 5 min in the presence of the examiner. For each bread, the
total volume of the test meal, as determined by mimicking chewing through homogenization of the
bread portion and 160 mL of water, was 215 mL (isovolumetric). On a di erent day, a standard liquid
test meal (Nutridrink®; Nutricia, Milano, Italy) was used as a reference to analyse gastrointestinal
symptoms and motility. It consisted of 200 mL liquid suspension containing 12 g (20%) protein, 11.6 g
(19%) fat, and 36.8 g (61%) carbohydrates for a total of 300 kcal, 1260 kJ and 455 mOsm /L. Results
obtained on Nutridrink in previous trials [ 38] also provide an internal validation tool and reference
kinetic for the in vivo postprandial markers of gastrointestinal function. Lactulose (10 g =15 mL
Lattulac®, SOFAR, Trezzano Rosa, Milan, Italy) was added to the reference meal [ 25]. The final volume
of the meal was 215 mL.
A randomized scheme (double blinded for breads) was used to administer the three types of bread
and the test meal to each subject. The meal administration to each volunteer was at three weeks-intervals.
2.7. Sensory Analysis, Perception of Appetite and Satiety, and Gastrointestinal Symptoms
A semi-quantitative scale (score 0–3) was used to record specific perceptions: appearance (crust
colour, attractiveness, porosity and elasticity); aroma (wheat, stale, mouldy, roasted and cereals);
taste (sweet, salty, bitter, sour, cereals and aftertaste); and texture (hardness, crustiness, chewiness,
adhesiveness and greasiness) [ 39], aiming at defining the peculiar organoleptic profiles of the breads.
Before each assay, the mouth was washed with plain water. Food, drink, smoking and physical activity
were prohibited before and during the assay. A quantitative Visual Analogue Scale (VAS 0–100 mm on
a horizontal line) was used to mark the degree of pleasantness of overall appearance, aroma, taste
and texture.
Detailed information was recorded from all volunteers regarding essential symptoms, which
eventually occurred the prior three months (e.g., feeling of abdominal fullness, epigastric pain, nausea
and/or vomiting, heartburn and additional gastrointestinal symptoms). Each symptom was scored
semi-quantitatively on the 0–3-point scale for severity, frequency and duration (Table 1). The VAS
monitored symptoms, together with the perception of appetite and satiety, at 30 min-intervals during
120 min following the ingestion of the test meal. VAS time-related curves for perception of appetite
and satiety and gastrointestinal symptoms, according to previous studies from our group [ 38,40]. The
corresponding Area Under Curve (AUC) were calculated through the software NCSS10 (NCSS LLC,
Kaysville, UT, USA).

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Table 1. Clinical characteristics of enrolled volunteers.
n 36
Males:Females 18:18
Age years (range) 25 1.1 (20–31)
BMI, kg /m2(range) 22.40.9 (18.6–29.4)
Fullness (severity) * 0.8 0.2
Fullness (frequency) ** 0.7 0.2
Fullness (duration) *** 0.7 0.2
Epigastric pain (severity) 0.9 0.4
Epigastric pain (frequency) 0.6 0.3
Epigastric pain (duration) 0.6 0.3
Nausea /vomiting (severity) 0.2 0.2
Nausea /vomiting (frequency) 0.2 0.1
Nausea /vomiting (duration) 0.6 0.3
Heartburn (severity) 0.5 0.2
Heartburn (frequency) 0.5 0.2
Heartburn (duration) 0.4 0.2
BMI, body mass index; data are expressed as mean SEM; Severity, frequency and duration are expressed on a 0–3
semiquantitative score (* 0 =absent; 1 =mild; 2 =moderate; 3 =severe; ** 0 =none; 1 =sometime; 2 =often; 4 =
interference with daily activities; *** 0 =none; 1 =less than 30 min daily; 2 =between 30 min and 2 h daily; 3 =more
than 2 h /daily).
2.8. Gallbladder and Gastric Motility
Gallbladder and gastric motility were determined simultaneously after meal
ingestion [25,26,38,41–46] . Time-dependent changes of fasting and postprandial gallbladder
volumes (mL) and antral areas (cm2) were calculated from frozen sonograms using a Noblus
ultrasound (Hitachi Medical, Tokyo, Japan) equipped with the 3.5 MHz convex transducer. Gallbladder
volume and antral area were measured before ( 5 and 0 min) and after meal ingestion, every 15–30 min
up to 120 min. Indices of gallbladder kinetics were fasting volume (mL) and residual volume
(minimum volume postprandial measured in mL and percent of fasting volume). Indices of gastric
emptying were antral (basal) area (cm2), maximal postprandial antral area (recorded after 5 min from
meal ingestion), and postprandial and minimal postprandial antral areas during the whole emptying
curve. Postprandial areas were also normalized to maximal areas after subtraction of basal areas
Normalized Postprandial Area =100(AtAbas)/(AmaxAbas), (3)
where A t=postprandial area at any given time; A bas=basal area; and A max=maximal antral area.
For both gallbladder and stomach, further indices included the Area Under the emptying Curve (AUC
expressed as mL120 min) and the half-emptying time (T 1/2, min). In particular, T 1/2was calculated
by linear regression analysis from the linear part of the emptying curves, thus corresponding to the
time in which 50% decrease of gallbladder volume and antral area were observed.
2.9. Oro-Cecal Transit Time
The OCTT was determined at the same time of gallbladder and gastric motility, using the H 2
breath technique, according to standard guidelines [ 37,38,42,47–50]. Samples of expired air were taken
before meal and subsequently, every 10 min during 180 min after meal ingestion. A pre-calibrated,
portable hydrogen sensitive electrochemical device (EC60-Gastrolyzer; Bedfont Scientific, Medford,
NJ, USA) was used to measure the time-dependent changes of H 2in breath, as a marker of cecal
fermentation of the unabsorbed lactulose. Results were expressed as H 2-excretion in parts per million
(ppm). The accuracy of the detector was 2 ppm. An increase of 10 ppm above the baseline for two
consecutive measurements was the OCTT, and calculated in min.

Nutrients 2019 ,11, 2954 8 of 21
2.10. Blood Analyses
To assess the impact of the test meal on glucose and amino acid levels, plasma samples (2 mL) were
taken from venous blood of volunteers at time 0 (baseline in the fasting state) and at 30 min-intervals
during 120 min after meal ingestion. For serum glucose analysis, 1 drop of blood (ca. 1 L) was
assayed using the Accu-Chek Active Meter (Roche Diagnostics, Indianapolis, Indiana, USA), and a
reflectometric glucose meter with plasma-corrected results. For amino acid analysis, venous blood was
collected in EDTA monovettes, and plasma was immediately separated by centrifugation (4000 rpm
for 8 min at 4C) and stored at20C. Total free amino acids were determined through the Biochrom
30 series Amino Acid Analyzer [19,51].
2.11. Statistical Analysis
Data from biochemical analyses were subject to one-way Analysis of Variance (ANOVA), using
the IBM SPSS Statistics 26 (IBM Corporation, New York City, NY, USA) software. Data from the
in vivo challenge were subjected to the ANOVA followed by the post-hoc comparison test., through
the software NCSS10 (NCSS LLC, Kaysville, UT, USA). Motility parameters data were subjected to
ANOVA, followed by the Tukey–Kramer Multiple-Comparison Test. A two-sided probability ( p<0.05)
was considered statistically significant.
3. Results
3.1. Semi-Industrial Manufacture of Sourdough and Baker’s Yeast Breads
The traditional and most di used protocol for making type-I sourdough (S) was used [ 20]. The
selection of well-known and well-characterized species of sourdough lactic acid bacteria and yeasts
ensured the reproducibility of the performance for bread making. Following daily refreshments, S
became mature. After the last fermentation (8 h), it reached the value of pH of 3.89 0.02 (Table 2). S
was used to prepare two di erent sourdoughs. The values of pH of these sourdoughs agreed with
the duration of their time of incubation. S 24was more acidic than S 4. Values of TTA had an opposite
trend. The cell density of lactic acid bacteria of both the sourdoughs exceeded the level of 9.0 log
cfu/g. The cell numbers of S 24were significantly ( p<0.05) higher than that of S 4. The cell density
of yeasts was also the highest in S 24. S had a concentration of lactic acid of 43.3 0.3 mmol /kg. In
comparison, the content of lactic acid in S 4was ca. 35% lower and that of S 24was more than twice.
Almost the same di erences were found for the concentration of acetic acid. The FQ ranged from
4.3 to 4.6, without significant ( p>0.05) di erences among the sourdoughs. All the biochemical and
microbiological data [ 20,21] proved that we produced two traditional sourdoughs (S 4and S 24), having
almost the same microbial load but di erent acidifying capabilities.
Table 2. Biochemical and microbiological characteristics of sourdoughs (dough yield of 160) used for
bread making.
S S 4 S24
pH 3.890.02b4.260.01a3.550.01c
TTA Total titratable acidity
(mL NaOH 0.1M /10g)6.600.11b3.200.09c9.000.15a
LAB (Log cfu /g) 9.530.13b9.180.10b9.750.08a
Yeasts (Log cfu /g) 7.330.05b7.070.07c7.500.05a
Lactic acid (mmol /kg) 43.30.3b28.20.2c93.30.3a
Acetic acid (mmol /kg) 10.20.2b6.20.2c20.30.2a
Fermentation Quotient (FQ) 4.3 0.2 4.5 0.2 4.6 0.2
Total Free Amino Acids (g /kg) 0.7220.018c1.3800.024b7.1740.032a
S, type-I sourdough, which was mature after four refreshments; S 4and S 24, sourdoughs produced by mixing S with
wheat flour and water, and fermented at 30C for 4 and 24 h, respectively.a–cValues in the same row with di erent
superscript letters di er significantly ( p<0.05).

Nutrients 2019 ,11, 2954 9 of 21
Two sourdough breads (sourdough bread, SB; and traditional sourdough bread, t-SB) and a
baker’s yeast bread (BYB) were manufactured at a semi-industrial scale. BYB had the highest value
of pH (5.60.1) (Table 3). Proximal composition of the breads (Supplementary Table S1) did not
show ( p>0.05) significant di erences among macronutrients. Gluten in BYB was 8.9 0.2%, and not
significant ( p>0.05) di erences were found for SB (8.7 0.3%). A significant ( p<0.05) lower gluten
amount was found in t-SB (7.6 0.2%). It was previously observed that proteolysis by sourdough
lactic acid bacteria on gluten proteins causes the release of soluble degradation products [18].
Table 3. Chemical, technological, and nutritional characteristics of the three types of bread.
BYB SB t-SB
Dough (before baking)
pH 5.6 0.1a4.90.1b4.40.1c
TTA (mL NaOH 0.1m /10g) 3.0 0.1c5.00.2b6.90.2a
Lactic acid (mmol /kg) 1.7 0.1c7.20.1b26.40.3a
Acetic acid (mmol /kg) nd 1.40.1b5.80.1a
FQ nd 4.20.1b4.50.2a
Total FAA (g /kg) 0.70 0.02c1.330.02b1.710.02a
Bread
Volume increase (%) 2315b2457a2234c
Specific volume (cm2/g) 3.30.1b3.60.1a2.90.1c
Textural parameters
Hardness (g) 321010b315021c347211a
Resilience 0.85 0.02a0.810.05a0.720.02b
Fracturability (g) 3075 5a295610b228210c
Image analysis
Black pixel area (%) 44.01.8b52.62.2a44.41.5b
Color analysis
L 60.2 1.6a60.40.7a54.70.8b
A 10.40.8b9.30.6b11.70.2a
B 35.7 1.0a34.10.2a33.60.5b
DE 48.11.7b46.60.6b51.30.6a
Nutritional indexes
In vitro protein digestibility
(IVPD, %)63.71.2c71.61.1b79.81.4a
Limiting Amino AcidsLysine
Methionine
TryptophanLysine
Methionine
TryptophanLysine
Methionine
Tryptophan
Protein score (%) 18.2 0.2c24.20.2b59.20.5a
Essential amino acids Index
(EAAI)43.20.4c56.30.5b72.30.5a
Biological value (BV) 35.4 0.3c39.70.3b54.10.4a
Protein E ciency Ratio
(PER)21.10.2c23.50.2b53.50.3a
Nutritional Index (NI) 2.8 0.1c3.40.1b5.50.1a
BYB, baker ´s yeast bread leavened with baker’s yeast (1.5%, w/w) for 90 min at 30C; SB, sourdough bread leavened
with S 4(20%, w/w) and baker’s yeast (1.5%, w/w) for 90 min at 30C; t-SB, sourdough bread leavened with S 24(20%,
w/w) for 4 h at 30C.a–cValues in the same row with di erent superscript letters di er significantly ( p<0.05); nd:
not detected.
As expected, the acidifying activity of sourdough lactic acid bacteria during dough fermentation
caused a marked decrease of the pH. t-SB had pH of 4.4 0.1, while SB, which was fermented with S 4
for a shorter time (1.5 vs. 4 h), had a significantly ( p<0.05) higher value of pH (4.9 0.1). In agreement,
the concentration of lactic acid of BYB was very low (1.7 0.1 mmol /kg). The content of lactic acid of
t-SB was ca. four-times higher than that found in SB. Acetic acid was found in both sourdough breads,
being significantly ( p<0.05) higher in t-SB. The values of FQ were 4.2 0.1 and 4.50.2 for SB and

Nutrients 2019 ,11, 2954 10 of 21
t-SB, respectively. Total FAA were found at the lowest level in BYB. The concentrations of FAA of SB
and t-SB were, respectively, ca. two- and two and half-times higher.
The di erent total FAA concentration can be considered as an index of the degree of proteolysis of
the breads, while the calculation of the protein concentration based on the organic nitrogen (Kjeldahl
method) did not provide information about the degradation status of the proteins, therefore resulting
similar for the three samples (Table S1).
During fermentation, the volume of all breads increased (ca. three-times) (Table 2). Compared to
BYB, the increase for SB was slightly but significantly ( p<0.05) higher. The lowest volume increase
was that of t-SB. In agreement, the specific volume and the gas cells area were the highest and the
lowest for SB and t-SB, respectively. Intermediate were the values of BYB. SB also showed the lowest
value of hardness. t-SB had the highest hardness and the lowest fracturability, which corresponded
to a remarkable friability of the crumb. The instrumental colour analysis indicated small di erences
for lightness (L) and colorimetric coordinates between BYB and SB. t-SB had the lowest and highest
values of L and DE, respectively. All biochemical and textural data proved that we manufactured
representative sourdough and baker‘s yeast breads, with di erences between sourdough breads (SB
and t-SB) that mimicked regional traditions [20,21].
3.2. In Vitro Protein Digestibility
The value of IVPD for BYB was ca. 63.7% (Table 3). Overall, the sourdough fermentation led to an
increase of the in vitro protein digestibility: ca. 8 and 16% for SB and t-SB, respectively. Lys, Met and
Trp were the limiting amino acids of the three types of bread. Nevertheless, the protein score was the
highest for SB and t-SB. Similarly, the values of EAAI, BV and PER, which are used to estimate the
quality of food proteins, were significantly ( p<0.05) higher in SB with respect to BYB. t-SB had the
highest values of these indices. The Nutritional Index (NI) calculation referred to both the amount of
digestible protein fraction and the ratio of essential amino acids. SB and, especially, t-SB had values of
this index ca. 21 and 62% higher than that determined for BYB. As documented in the literature [ 18,52],
we confirmed that the in vitro protein digestibility improves with sourdough fermentation.
3.3. Breads Sensory Profile and Perception of Appetite, Satiety, and Gastrointestinal Symptoms
Volunteers used questionnaires to describe the sensory properties of the three types of bread.
They explicited attributes for appearance, aroma, taste and texture in a semi-quantitative scale. No
significant ( p>0.05) di erences were found for attributes of appearance, such as attractiveness and
elasticity (Figure 1A). Only the crust colour and porosity of SB were scored as significantly ( p<0.05)
the highest. The scores for stale and mouldy aroma were low for all the three types of bread. Wheat
aroma primarily connoted BYB. The aroma of SB was described as roasted and cereals. Bitter and sour
taste mainly distinguished t-SB, with the same attributes that received significantly ( p<0.05) lower
scores for SB. Overall, the aftertaste of both the sourdough breads was the most intense. Volunteers
did not perceive significant ( p>0.05) di erences for hardness, crustiness and greasiness among the
three types of bread. Chewiness and adhesiveness scores were the highest in BYB. Volunteers assessed
the bread pleasantness according to the Visual Analogic Scale (VAS) approach. The overall taste and
texture did not significant ( p>0.05) di er (Figure 1B).
Compared to the other two types of bread, the overall appearance and aroma were significantly
(p<0.05) higher for SB. Using VAS approach, appetite and satiety, and the gastrointestinal symptoms
were monitored during 120 min following ingestion. After 30 min, SB and t-SB stimulated more
appetite than BYB (Figure 2A).

Nutrients 2019 ,11, 2954 11 of 21
Nutrients 2019 , 11, x FOR PEER REVIEW 10 of 20
BYB, baker´s yeast bread leavened with baker’s yeast (1.5%, w/w) for 90 min at 30 °C; SB, sourdough
bread leavened with S 4 (20%, w/w) and baker’s yeast (1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough
bread leavened with S 24 (20%, w/w) for 4 h at 30 °C. a–c V a l u e s i n t h e s a m e r o w w i t h d i f f e r e n t
superscript letters differ significantly ( p < 0.05); nd: not detected.
3.2. In Vitro Protein Digestibility
The value of IVPD for BYB was ca. 63.7% (Table 3). Overall, the sourdough fermentation led to
an increase of the in vitro protein digestibility: ca. 8 and 16% for SB and t-SB, respectively. Lys, Met and Trp were the limiting amino acids of the three types of bread. Nevertheless, the protein score was the highest for SB and t-SB. Similarly, the values of EAAI, BV and PER, which are used to estimate
the quality of food proteins, were significantly ( p < 0.05) higher in SB with respect to BYB. t-SB had
the highest values of these indices. The Nutritional Index (NI) calculation referred to both the amount
of digestible protein fraction and the ratio of essential amino acids. SB and, especially, t-SB had values
of this index ca. 21 and 62% higher than that dete rmined for BYB. As documented in the literature
[18,52], we confirmed that the in vitro protein digestibility improves with sourdough fermentation.
3.3. Breads Sensory Profile and Perception of Ap petite, Satiety, and Gast rointestinal Symptoms
Volunteers used questionnaires to describe the se nsory properties of the three types of bread.
They explicited attributes for appearance, aroma, taste and texture in a semi-quantitative scale. No significant ( p > 0.05) differences were found for attributes of appearance, such as attractiveness and
elasticity (Figure 1A). Only the crust colour and porosity of SB were scored as significantly ( p < 0.05)
the highest. The scores for stale and mouldy aroma were low for all the three types of bread. Wheat aroma primarily connoted BYB. The aroma of SB was described as roasted and cereals. Bitter and sour taste mainly distinguished t-SB, with the same attributes that received significantly ( p < 0.05)
lower scores for SB. Overall, the aftertaste of both the sourdough breads was the most intense.
Volunteers did not perceive significant ( p > 0.05) differences for hard ness, crustiness and greasiness
among the three types of bread. Chewiness and adhesiveness scores were the highest in BYB.
Volunteers assessed the bread pleasantness according to the Visual Analogic Scale (VAS) approach.
The overall taste and texture did not significant ( p > 0.05) differ (Figure 1B).

Figure 1. Sensory analysis of the three types of bread. BYB, baker´s yeast bread leavened with baker’s
yeast (1.5%, w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s
yeast (1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at
30°C. ( A) spider web chart of the perception scores (semi-quantitative scale 0–3); ( B) degree of
pleasantness (visual analogue scale, VAS, 0–100 mm). a–c Within the same parameter, values with
different superscript lette rs differ significantly ( p < 0.05). 0123Crust color
Attractiveness
Porosity
Elasticity
Wheat
Stale
Mouldy
Rosted
Cereals
Sweet
SaltyBitterSourCerealAftertasteHardnessCrustinessChewinessAdhedivenessGreasiness
BYB
SB
SBT
AromaAppearence
TasteTexture
Adhesiveness
RoastedA B
t-SB
010203040506070
Appearence Aroma Taste TextureVAS (mm)
BYB SB t-SBaa
a a
aaa
abb
bb
Figure 1. Sensory analysis of the three types of bread. BYB, baker ´s yeast bread leavened with baker’s
yeast (1.5%, w/w) for 90 min at 30C; SB, sourdough bread leavened with S 4(20%, w/w) and baker’s
yeast (1.5%, w/w) for 90 min at 30C; t-SB, sourdough bread leavened with S 24(20%, w/w) for 4 h
at 30C. (A) spider web chart of the perception scores (semi-quantitative scale 0–3); ( B) degree of
pleasantness (visual analogue scale, VAS, 0–100 mm).a–cWithin the same parameter, values with
dierent superscript letters di er significantly ( p<0.05).
Nutrients 2019 , 11, x FOR PEER REVIEW 11 of 20
Compared to the other two types of bread, the overall appearance and aroma were significantly
(p < 0.05) higher for SB. Using VAS approach, appetite and satiety, and the gastrointestinal symptoms
were monitored during 120 min following ingestion. After 30 min, SB and t-SB stimulated more
appetite than BYB (Figure 2A).

Figure 2. Perception of appetite and satiety ( A), and gastrointestinal symptoms (nausea and fullness)
(B) in response to ingestion of the test meals. Time-dependent changes were scored with Visual
Analogic Scale (VAS, 0–100 mm), an d represented as mean ± SEM (Standard Error of the Mean) and
area under curve (AUC). BYB, baker´s yeast bread leavened with baker’s yeast (1.5%, w/w) for 90 min
at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s yeast (1.5%, w/w) for 90 min
at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at 30 °C. NU, Nutridrink used as
the reference. a−c Within the same parameter, values wi th different superscript letters differ
significantly ( p < 0.05).
After 60 min, no significant ( p > 0.05) differences were found among breads. Sixty minutes after
ingesting Nutridrink (NU), the significantly ( p < 0.05) lowest Area Under Curve (AUC) indicated the
lowest perception of appetite. After 120 min from ingestion, the highest satiety perception was associated with NU consumption. Comparing breads , the ingestion of t-SB was associated with the
curve having the significantly ( p < 0.05) highest AUC. No symptoms of nausea were perceived (scores
always lower than 10 mm) after ingesting breads (Figure 2B). Significantly ( p < 0.05) higher scores
were reported after NU ingestion. The consumptio n of NU also associated to the lowest fullness
perception. AUC for fullness were similar among breads. Nevertheless, t-SB showed the highest score
at 30 min. Volunteers did not report epigastric pain after ingesting breads or NU. In particular, VAS
scores were lower than 15 mm during the 120 min following the samples ingestion. AUC was similar
(p > 0.05) and lower than 1000 for all the breads and NU.
3.4. Gallbladder and Gastric Motilit y and Determination of the OCTT
Volunteers showed similar fasting gallbladder volumes across the four days of challenging (17.6
± 1.2 – 18.9 ± 1.3 mL) (Figure 3A and Supplementary Table S2). Time (min)0 3 06 09 0 1 2 0Appetite VAS (mm)
020406080100
Time (min)0 3 06 09 0 1 2 0Satiety VAS (mm)
020406080100
0200040006000800010000
B Y BS Bt – S B N USatiety AUCa
bb
cBYB
SB
t-SB
NU0200040006000800010000
BYB SB t-SB NUAppetite AUCa
b b
c
Time (min)0 2 04 06 08 0 1 0 0 1 2 0Fullness VAS (mm)
020406080100
0200040006000800010000
BYB SB t-SB NUFullness AUC0200040006000800010000
B Y BS Bt – S B N UNausea AUC
a
ba
aBYB
SB
t-SB
NUa
b bb
Time (min)0 3 06 09 0 1 2 0Nausea V AS (mm)
020406080100BA
Figure 2. Perception of appetite and satiety ( A), and gastrointestinal symptoms (nausea and fullness)
(B) in response to ingestion of the test meals. Time-dependent changes were scored with Visual Analogic
Scale (VAS, 0–100 mm), and represented as mean SEM (Standard Error of the Mean) and area under
curve (AUC). BYB, baker ´s yeast bread leavened with baker’s yeast (1.5%, w/w) for 90 min at 30C; SB,
sourdough bread leavened with S 4(20%, w/w) and baker’s yeast (1.5%, w/w) for 90 min at 30C; t-SB,
sourdough bread leavened with S 24(20%, w/w) for 4 h at 30C. NU, Nutridrink used as the reference.
a–cWithin the same parameter, values with di erent superscript letters di er significantly ( p<0.05).
After 60 min, no significant ( p>0.05) di erences were found among breads. Sixty minutes after
ingesting Nutridrink (NU), the significantly ( p<0.05) lowest Area Under Curve (AUC) indicated
the lowest perception of appetite. After 120 min from ingestion, the highest satiety perception was
associated with NU consumption. Comparing breads, the ingestion of t-SB was associated with the
curve having the significantly ( p<0.05) highest AUC. No symptoms of nausea were perceived (scores
always lower than 10 mm) after ingesting breads (Figure 2B). Significantly ( p<0.05) higher scores were
reported after NU ingestion. The consumption of NU also associated to the lowest fullness perception.
AUC for fullness were similar among breads. Nevertheless, t-SB showed the highest score at 30 min.

Nutrients 2019 ,11, 2954 12 of 21
Volunteers did not report epigastric pain after ingesting breads or NU. In particular, VAS scores were
lower than 15 mm during the 120 min following the samples ingestion. AUC was similar ( p>0.05)
and lower than 1000 for all the breads and NU.
3.4. Gallbladder and Gastric Motility and Determination of the OCTT
Volunteers showed similar fasting gallbladder volumes across the four days of challenging (17.6
1.218.91.3 mL) (Figure 3A and Supplementary Table S2).
Nutrients 2019 , 11, x FOR PEER REVIEW 12 of 20

Figure 3. Gastric ( A) and gallbladder ( B) emptying curves in response to the ingestion of the test meals
(isovolumetric). Time-dependent changes of antral area (cm2) are represented as mean ± SEM
(Standard Error of the Mean) and area under curv e (AUC). Time-dependent changes of gallbladder
volume (mL) are represented as mean ± SEM and ar ea under curve (AUC). BY B, baker´s yeast bread
leavened with baker’s yeast (1.5%, w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4
(20%, w/w) and baker’s yeast (1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24
(20%, w/w) for 4 h at 30 °C. NU, Nutrid rink used as the reference. a–b Within the same parameter,
values with different superscrip t letters differ significantly ( p < 0.05).
The ingestion of the three types of bread indu ced similar responses. In detail, the mean
gallbladder ejection volume ranged from 35.5 to 43.5% (mean residual volume of ca. 11 ml), the half-
emptying time varied from 26.7 ± 2.3 to 33.6 ± 2.8 min, and the half-refilling rate was 0.1 ± 0.0 ml/min. Compared to breads, NU induced a faster gallbladder emptying and refilling. This was confirmed by significantly ( p < 0.05) different kinetic parameters (ejectio n volume of 68.5 ± 3.1%; residual volume
of 5.9%; half-emptying time of 21.9 ± 1.4 min; and ha lf-refilling rate of 2.4 ± 0.4 ml/min). At baseline,
gastric basal antral area ranged from 4.0 ± 0.2 to 4.3 ± 0.3 cm
2 (Figure 3B). Within five minutes after
ingestion, the antral area reached the maximum postprandial area (10.7 ± 0.4 − 11.7 ± 0.5 cm2), without
any significant ( p > 0.05) difference among breads. The inge stion of different meals corresponded to
different time-related decreases of the antral area. In particular, the emptying rate corresponding to
SB and t-SB ingestion was significantly ( p < 0.05) lower than that of BYB ( −0.24 ± 01 and −0.28 ± 0.1 vs.
−1.20 ± 0.1 cm2/min, Supplementary Table S2). Accordingly, the half-emptying time of BYB (43.7 ± 4.4
min) was significantly ( p < 0.05) longer than that found for SB and t-SB (34.8 ± 2.4 and 30.8 ± 2.3 min,
respectively). The AUC amplitude for BYB was the highest (854 ± 119 vs . 774 ± 105 and 758 ± 105 cm2
× 120 min for SB and t-SB, respectively) (Supplementa ry Table S2). Response to NU ingestion showed
that the gastric half-emptying time was markedly and significantly ( p < 0.05) lower than that of BYB.
This probably because of the different viscosity after chewing and ingestion. Nevertheless, it was comparable to both sourdough breads, having crumbly texture and lower fracturability than BYB. In detail, the NU ingestion was characterized by emptying rate, half-emptying time and AUC of −0.66 0200040006000800010000
BYB SB t-SB NUAUC
bb ba02004006008001000
B Y BS Bt – S B N UAUCa
a a
a
Time (min)-5 0 5 1015202530 45 60 75 90 105 120Antral area (cm2)
456789101112
Time (min)- 5 0 1 53 04 56 07 59 0 1 0 5 1 2 0Gallbladder Volume (%)
0102030405060708090100
BYB
SB
t-SB
NUBA
Figure 3. Gastric ( A) and gallbladder ( B) emptying curves in response to the ingestion of the test
meals (isovolumetric). Time-dependent changes of antral area (cm2) are represented as mean SEM
(Standard Error of the Mean) and area under curve (AUC). Time-dependent changes of gallbladder
volume (mL) are represented as mean SEM and area under curve (AUC). BYB, baker ´s yeast bread
leavened with baker’s yeast (1.5%, w/w) for 90 min at 30C; SB, sourdough bread leavened with S 4
(20%, w/w) and baker’s yeast (1.5%, w/w) for 90 min at 30C; t-SB, sourdough bread leavened with
S24(20%, w/w) for 4 h at 30C. NU, Nutridrink used as the reference.a–bWithin the same parameter,
values with di erent superscript letters di er significantly ( p<0.05).
The ingestion of the three types of bread induced similar responses. In detail, the mean gallbladder
ejection volume ranged from 35.5 to 43.5% (mean residual volume of ca. 11 mL), the half-emptying time
varied from 26.72.3 to 33.62.8 min, and the half-refilling rate was 0.1 0.0 mL /min. Compared to
breads, NU induced a faster gallbladder emptying and refilling. This was confirmed by significantly
(p<0.05) di erent kinetic parameters (ejection volume of 68.5 3.1%; residual volume of 5.9%;
half-emptying time of 21.9 1.4 min; and half-refilling rate of 2.4 0.4 mL /min). At baseline, gastric
basal antral area ranged from 4.0 0.2 to 4.30.3 cm2(Figure 3B). Within five minutes after ingestion,
the antral area reached the maximum postprandial area (10.7 0.411.70.5 cm2), without any
significant ( p>0.05) di erence among breads. The ingestion of di erent meals corresponded to
dierent time-related decreases of the antral area. In particular, the emptying rate corresponding to

Nutrients 2019 ,11, 2954 13 of 21
SB and t-SB ingestion was significantly ( p<0.05) lower than that of BYB ( 0.2401 and0.280.1
vs.1.200.1 cm2/min, Supplementary Table S2). Accordingly, the half-emptying time of BYB (43.7
4.4 min) was significantly ( p<0.05) longer than that found for SB and t-SB (34.8 2.4 and 30.8
2.3 min, respectively). The AUC amplitude for BYB was the highest (854 119 vs. 774105 and 758
105 cm2120 min for SB and t-SB, respectively) (Supplementary Table S2). Response to NU ingestion
showed that the gastric half-emptying time was markedly and significantly ( p<0.05) lower than that
of BYB. This probably because of the di erent viscosity after chewing and ingestion. Nevertheless,
it was comparable to both sourdough breads, having crumbly texture and lower fracturability than
BYB. In detail, the NU ingestion was characterized by emptying rate, half-emptying time and AUC of
0.660.0, 29.01.9 min and 76519 cm2120 min, respectively. The OCTT was assessed through
the H 2breath technique. Volunteers consumed lactulose together with breads and the fermentation
of this indigestible sugar by colon microbes produced H 2. The concentration of H 2in breath was
measured every 10 min after meal ingestion. The time needed to exceed the baseline value (10 ppm)
was determined and considered as the entrance point of the food bolus into the large intestine, which
corresponds to OCTT. The OCTT median value for BYB was 89.5 min, which was not significantly
(p>0.05) di erent than that found for SB (80.5 min). On the contrary, this value was markedly and
significantly ( p<0.05) longer than that of t-SB (69.5 min) (Figure 4A,B).
Nutrients 2019 , 11, x FOR PEER REVIEW 13 of 20
± 0.0, 29.0 ± 1.9 min and 765 ± 19 cm2 × 120 min, respectively. The OCTT was assessed through the H 2
breath technique. Volunteers cons umed lactulose together with brea ds and the fermentation of this
indigestible sugar by colon microbes produced H 2. The concentration of H 2 in breath was measured
every 10 min after meal ingestion. The time ne eded to exceed the baseline value (10 ppm) was
determined and considered as the entrance point of the food bolus into the large intestine, which
corresponds to OCTT. The OCTT median value for BY B was 89.5 min, which was not significantly ( p
> 0.05) different than that found for SB (80.5 min). On the contrary, this value was markedly and significantly ( p < 0.05) longer than that of t-SB (69.5 min) (Figure 4A,B).

Figure 4. Oro-cecal transit time (OCTT) in response to the ingestion of test meals. ( A) time-dependent
curves of H2 concentration (ppm) in exhaled air, expressed as mean ± SEM (Standard Error of the
Mean); ( B) OCTT (min); ( C) H2 peak. BYB, baker´s yeast bread leavened with baker’s yeast (1.5%,
w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s yeast (1.5%,
w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at 30 °C. NU,
Nutridrink used as the reference. a–c Within the same parameter, va lues with different superscript
letters differ significantly ( p < 0.05).
The OCTT (median value 103.2 ± 3.4 min) of NU was significantly ( p < 0.05) the lowest. The H 2
peak for BYB was up to 30% higher ( p < 0.05) compared to both sourdough breads and NU (Figure
4C).
3.5. Postprandial Gl ycaemia and Free Amino Acids Absorption
Glycaemia was assessed by analysing the concentration of glucose from blood samples that were
collected after meal ingestion. All breads showed peaks of serum glucose concentration from 40 to 60
min after ingestion. The peak following BYB ingestion corresponded to 14.3 ± 0.39 mg/mL (Figure
5A), which was ca. 5% lower ( p < 0.05) than those reported for SB and t-SB. Compared to BYB, SB and
t-SB generated a lower glyc aemic curve (Figure 5B). 020406080100120
BYB SB t-SB NUOCTT (min)b
cba
Time (min)0 1 02 03 04 05 06 07 08 09 0 1 0 0 1 1 0 1 2 0H2 (ppm)
05101520253035BYB
SB
t-SB
NU2
051015202530
B Y BS Bt – S B N UPeak H2(ppm)a
b b
bA B
C
Figure 4. Oro-cecal transit time (OCTT) in response to the ingestion of test meals. ( A) time-dependent
curves of H2 concentration (ppm) in exhaled air, expressed as mean SEM (Standard Error of the
Mean); ( B) OCTT (min); ( C) H2 peak. BYB, baker ´s yeast bread leavened with baker’s yeast (1.5%, w/w)
for 90 min at 30C; SB, sourdough bread leavened with S 4(20%, w/w) and baker’s yeast (1.5%, w/w) for
90 min at 30C; t-SB, sourdough bread leavened with S 24(20%, w/w) for 4 h at 30C. NU, Nutridrink
used as the reference.a–cWithin the same parameter, values with di erent superscript letters di er
significantly ( p<0.05).
The OCTT (median value 103.2 3.4 min) of NU was significantly ( p<0.05) the lowest. The H 2
peak for BYB was up to 30% higher ( p<0.05) compared to both sourdough breads and NU (Figure 4C).
3.5. Postprandial Glycaemia and Free Amino Acids Absorption
Glycaemia was assessed by analysing the concentration of glucose from blood samples that were
collected after meal ingestion. All breads showed peaks of serum glucose concentration from 40
to 60 min after ingestion. The peak following BYB ingestion corresponded to 14.3 0.39 mg /mL

Nutrients 2019 ,11, 2954 14 of 21
(Figure 5A), which was ca. 5% lower ( p<0.05) than those reported for SB and t-SB. Compared to BYB,
SB and t-SB generated a lower glycaemic curve (Figure 5B).
Nutrients 2019 , 11, x FOR PEER REVIEW 14 of 20

Figure 5. Serum glucose concentration in response to the ingestion of the test meals. (A) time-
dependent curves of serum glucose (mg/dL) and ( B) area under curve (AUC). Data are expressed as
mean ± SEM (Standard Error of the Mean). BYB, baker´s yeast bread leaven ed with baker’s yeast
(1.5%, w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s yeast
(1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at 30 °C.
a–c Values with different superscr ipt letters differ significantly ( p < 0.05).
The AUC of BYB was 14947 ± 416 mg × 120 min, while the areas for SB and t-SB were 11 and 25%
lower ( p < 0.05), respectively. Before me al ingestion, the concentration of total FAA in the volunteer
blood plasmas ranged from 45 ± 5 to 60 ± 3 mg/L. This concentration increased after ingesting all breads, and reached the maximum peak at 60 min. At this time, the concentrations (median values)
were 145 ± 6, 152 ± 5 and 190 ± 5 mg/L for blood plasmas collected after ingesting BYB, SB and t-SB,
respectively (Figure 6A).

Figure 6. Serum total free amino acids (FAA) concentration in response to the ingestion of test meals.
Aggregated data are represented in box-plots: ( A) total FAA concentration at 60 min after ingestion;
(B) total FAA concentration at 120 min after ingestion. BYB, baker´s yeast bread leavened with baker’s
yeast (1.5%, w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s
yeast (1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at 30
°C. 020004000600080001000012000140001600018000
BYB SB t-SBAUCa
cbA B
Time (min)0 20 40 60 80 100 120 140Serum glucose (mg/dl)
8090100110120130140150
BYB
SB
t-SB
Figure 5. Serum glucose concentration in response to the ingestion of the test meals. ( A) time-dependent
curves of serum glucose (mg /dL) and ( B) area under curve (AUC). Data are expressed as mean SEM
(Standard Error of the Mean). BYB, baker ´s yeast bread leavened with baker’s yeast (1.5%, w/w) for
90 min at 30C; SB, sourdough bread leavened with S 4(20%, w/w) and baker’s yeast (1.5%, w/w) for
90 min at 30C; t-SB, sourdough bread leavened with S 24(20%, w/w) for 4 h at 30C.a–cValues with
dierent superscript letters di er significantly ( p<0.05).
The AUC of BYB was 14947 416 mg120 min, while the areas for SB and t-SB were 11 and 25%
lower ( p<0.05), respectively. Before meal ingestion, the concentration of total FAA in the volunteer
blood plasmas ranged from 45 5 to 603 mg /L. This concentration increased after ingesting all
breads, and reached the maximum peak at 60 min. At this time, the concentrations (median values)
were 1456, 1525 and 1905 mg /L for blood plasmas collected after ingesting BYB, SB and t-SB,
respectively (Figure 6A).
Nutrients 2019 , 11, x FOR PEER REVIEW 14 of 20

Figure 5. Serum glucose concentration in response to the ingestion of the test meals. (A) time-
dependent curves of serum glucose (mg/dL) and ( B) area under curve (AUC). Data are expressed as
mean ± SEM (Standard Error of the Mean). BYB, baker´s yeast bread leaven ed with baker’s yeast
(1.5%, w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s yeast
(1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at 30 °C.
a–c Values with different superscr ipt letters differ significantly ( p < 0.05).
The AUC of BYB was 14947 ± 416 mg × 120 min, while the areas for SB and t-SB were 11 and 25%
lower ( p < 0.05), respectively. Before me al ingestion, the concentration of total FAA in the volunteer
blood plasmas ranged from 45 ± 5 to 60 ± 3 mg/L. This concentration increased after ingesting all breads, and reached the maximum peak at 60 min. At this time, the concentrations (median values)
were 145 ± 6, 152 ± 5 and 190 ± 5 mg/L for blood plasmas collected after ingesting BYB, SB and t-SB,
respectively (Figure 6A).

Figure 6. Serum total free amino acids (FAA) concentration in response to the ingestion of test meals.
Aggregated data are represented in box-plots: ( A) total FAA concentration at 60 min after ingestion;
(B) total FAA concentration at 120 min after ingestion. BYB, baker´s yeast bread leavened with baker’s
yeast (1.5%, w/w) for 90 min at 30 °C; SB, sourdough bread leavened with S 4 (20%, w/w) and baker’s
yeast (1.5%, w/w) for 90 min at 30 °C; t-SB, sourdough bread leavened with S 24 (20%, w/w) for 4 h at 30
°C. 020004000600080001000012000140001600018000
BYB SB t-SBAUCa
cbA B
Time (min)0 20 40 60 80 100 120 140Serum glucose (mg/dl)
8090100110120130140150
BYB
SB
t-SB
Figure 6. Serum total free amino acids (FAA) concentration in response to the ingestion of test meals.
Aggregated data are represented in box-plots: ( A) total FAA concentration at 60 min after ingestion;
(B) total FAA concentration at 120 min after ingestion. BYB, baker ´s yeast bread leavened with baker’s
yeast (1.5%, w/w) for 90 min at 30C; SB, sourdough bread leavened with S 4(20%, w/w) and baker’s
yeast (1.5%, w/w) for 90 min at 30C; t-SB, sourdough bread leavened with S 24(20%, w/w) for 4 h at
30C.

Nutrients 2019 ,11, 2954 15 of 21
Although the concentrations of total FAA of both sourdough breads were higher, the di erences
among breads were not significant ( p>0.05). After 60 min, the concentrations of total FAA decreased
according to kinetics that varied depending on the type of bread (Figure 6B). At 120 min, the
concentration of total FAA of BYB was similar ( p>0.05) to the baseline value (decrease by 67%). On
the contrary, the decreases observed in the blood plasmas of volunteers who ingested SB and t-SB were
very low (17 and 10%, respectively).
4. Discussion
Scientific evidence has been gathered to prove the nutritional e ectiveness of the sourdough
fermentation [ 8,53] but investigations on bread digestibility are still too limited and partial. The
tradition and popular opinion converge on the alleged greater digestibility and lightness of long-time
fermented sourdough breads with respect to fast processing by chemicals or baker’s yeast. First, we
aimed at deepening the knowledge in this regard, combining wide-spectrum characterization of breads,
in vitro nutritional indices, and, primarily, in vivo postprandial markers from healthy volunteers.
All microbiological (e.g., cell densities and ratio between lactic acid bacteria and yeasts of 100:1)
and biochemical (e.g., pH, content of organic acids, FQ and concentration of total FAA) data supported
the peculiar features of baker´s yeast bread and two di erent types of sourdough breads [20].
A number of hedonistic attributes (e.g., palatability, aroma, texture, appearance and overall
pleasantness) markedly influence the consumer approach to a meal. These attributes relate to food
chemical and physical features, which have shaped during processing [ 38]. Sourdough fermentation
causes the appearance of distinctive sensory attributes in breads [7]. Salty, bitter and, especially, sour
tastes distinguished sourdough breads, with very low scores for chewiness and adhesiveness. As
usual for dietary studies, the assessment of pleasantness was through the VAS [ 13,54]. Reflecting
the current market trend and considering the relatively young age (20–31 years old) of volunteers,
SB, manufactured with a mild acidification, was preferred for aroma and appearance (Figure 1).
The most pronounced sour taste primarily attained to the old tradition. Together with the sensory
attributes, the perception of appetite, satiety and gastrointestinal symptoms is essential to assess food
digestibility because tightly correlated with gastric emptying and intestinal fermentation [ 13,14]. It
must be highlighted that the present study aimed at investigating bread digestibility in healthy subjects,
acting as reference group for further studies on subjects with upper GI symptoms such as bloating,
reflux, epigastric pain or irritable bowel syndrome. Absent or low intensity gastrointestinal symptoms,
including fullness, are associated to post-prandial well-being [ 13]. As expected, the ingestion of all
the three types of bread did not cause higher nausea and epigastric pain. SB, made with moderate
sourdough acidification, stimulated more the appetite. Within 60 min after ingestion, SB also induced
lower satiety and fullness perceptions (Figure 2). Other authors [ 13,55] described an inverse correlation
between appetite and satiety perceptions. t-SB, having the most intense acidic and salty taste, and the
most compact structure, induced the highest fullness perception in the shortest time (30 min). A
previous report [ 13] on croissants made with sourdough showed almost similar results, combining
lower appetite and higher satiety with respect to the consumption of croissants made with baker’s yeast.
NU is a commercial preparation with a defined nutrient composition and gastric and small intestinal
motility [ 41,42]. The gallbladder response did not di er among breads (Figure 3B), nevertheless, the
response to the isovolumetric NU was di erent. As expected, compared to breads, the ingestion of NU
caused a markedly higher ejection volume and refilling time, with a kinetic curve of the gallbladder
volume that determined an AUC ca. two-times higher. The higher content of lipids of NU might had
been responsible for these di erences.
Overall, the nutrient composition and quantity of the ingested food influence its stomach transit.
The alteration of the transit could induce fullness and nausea [ 13]. As estimated using paracetamol
adsorption or the13C-octanoic acid breath test, previous in vivo challenges [ 56,57] failed to show
dierences in gastric emptying after ingesting breads made with di erent cereal flours and leavening
agents. Through an ultrasonographic analysis at gastric level, we were able to show a faster emptying

Nutrients 2019 ,11, 2954 16 of 21
time for sourdough breads compared to BYB (Figure 3A). Sourdough breads had lower values of pH
and higher TTA than baker’s yeast bread, which led to the hypothesis of a quicker gastric acidification
of the bolus. The di erent status of the organic nitrogen fraction among breads might had played also
a relevant function. Contrary to yeasts, sourdough lactic acid bacteria have a more intense proteolytic
activity because of an e cient proteolytic system, comprising cell-wall associated proteinases, a wide
portfolio of intracellular peptidases and specific membrane transporters [ 8]. The progressive hydrolysis
of wheat naive proteins, including gluten, led to a consistent increase of peptides and free amino acids
during sourdough fermentation. Like in a process of pre-digestion, the occurring proteolysis increased
the amount of the in vitro digestible protein fraction (up to ca. 80%) as shown by several nutritional
indices (IVPD, EAAI, BV , PER and NI). The degree of proteolysis was proportional to the duration of
the fermentation. As analysed by magnetic resonance imaging, faster gastric emptying also followed
the ingestion of sourdough croissants with respect to those made with baker’s yeast [ 13]. All these
data contrasted previous findings of Liljeberg and Bjorck [ 58] that suggested a decrease of the gastric
emptying in presence of organic acids. The rapidity of the gastric emptying correlates with a reduced
perception of nausea, abdominal discomfort, fullness and satiety [ 59]. This correlation perfectly fits with
what observed for SB but disagreed with respect to the behaviour of t-SB, which had comparable gastric
emptying rate but caused a more persistent fullness and satiety perception. The hypothesis is that
sourdough baked goods did not induce a mechanical satiety but modulated the feelings of hunger and
satiety by stimulating the hormonal response through metabolically active compounds [ 13]. The type
and amount of proteins influence the gastric emptying time, and the satiety and appetite perceptions
through the modulated release of hormones, such as glucagon-like peptide (GLP)-1, oxyntomodulin,
pancreatic polypeptides and glucose-dependent insulin-trophic polypeptide (GIP) [ 60,61]. Although it
was demonstrated that an increased protein content delayed gastric emptying [ 60], no information on
the eect of hydrolysed proteins is currently available.
The administration of lactulose together with the test meal is one of the most e cient tools to
monitor the oro-cecal transit time through the estimation of the H 2level in breath [ 35]. The dose
of lactulose administered to volunteers is markedly lower than the amount previously associated
to the gastrointestinal motility increase and osmotic diarrhoea [ 62]. First, we showed a prolonged
transit time for the baker’s yeast bread and a faster passage of sourdough breads, especially when
made with traditional and long-time fermentation, whose transit lasted ca. 20 min less than BYB
(Figure 4). This finding was consistent with the results from gastric emptying. It was hypothesized a
dierent carbohydrate profile between baked goods made with baker’s yeast and those fermented
with sourdough. The diverse and complementary metabolic activities of the sourdough microbiota,
together with flour endogenous enzymes (e.g., - and -amylases, gluco-amylases), led to a substrate
composition that uniquely influenced the gas production by intestinal microbes [ 13]. An in vitro
study [ 63] demonstrated that the pattern of carbohydrates derived from sourdough fermentation
caused a low cumulative gas emission after 15 h of fermentation by the intestinal microbes.
Dierences in carbohydrate digestibility and absorption among breads determined di erent
post-prandial glycaemia responses (Figure 5). Sourdough breads had the lowest values. The glycaemic
index (GI) (ratio of the areas under the glycaemic curves between tested bread and reference white
bread, in this case BYB) was ca. 89 and 75 for SB and t-SB, respectively. These findings confirmed
other in vivo determinations [ 16,17,64]. Primarily, the decreased GI of sourdough breads correlated to
biological acidification [ 65] in presence of low values of pH (3.5–4.0), which promote the formation of
resistant starch [ 66]. Fibbers [ 16] and phenols [ 67] may have an e ect. During sourdough fermentation,
lactic acid bacteria have the capability to increase the fiber soluble fraction and the concentration of
free phenolic compounds [ 8,52]. Nevertheless, because of the low concentration of fibres and phenols
in the white wheat flour used for bread making, a weak contribution is conceivable. Because the
relevant di erence for proteolysis between baker’s yeast and sourdough breads, we innovatively
estimated the total FAA absorption as a further measurement of the bread digestibility. The total
concentration of FAA in blood plasmas increased following the ingestion of all breads. After ingestion

Nutrients 2019 ,11, 2954 17 of 21
of sourdough breads, having a concentration of total FAA markedly higher than that of BYB, the levels
of these nitrogen derivatives in blood plasmas maintained high and almost constant for extended time
(Figure 6).
5. Conclusions
As a staple food, information on bread digestibility deserve a marked interest from a nutritional
perspective. Bread is the generic name for a group of leavened food products, manufactured with a wide
range of flours and using di erent leavening processes. The traditional and popular opinion concerns
a high digestibility of long-time fermented breads although confirmatory scientific data are lacking.
The present study presents some limitations: (i) only healthy normal subjects (without gastrointestinal
symptoms) were enrolled; (ii) questionnaires for gastrointestinal symptoms investigation are not largely
validated (iii) H 2-breath test (with lactulose) is not the gold-standard method to asses gastrointestinal
transit, however it is non-invasive, well accepted by subjects (radiations or intubations are not required),
and largely used to assess the OCTT. Nevertheless, we combined wide-spectrum characterization of
breads, in vitro nutritional indices, and in vivo postprandial markers, which all agreed and somewhat
explained the presumed better digestibility of sourdough compared to baker’s yeast breads. Moreover,
a dierent clinical response to sourdough breads, representative of the most common production
protocols and characterized by di erent levels of acidification and proteolysis, was highlighted.
Supplementary Materials: The following are available online at http: //www.mdpi.com /2072-6643 /11/12/2954/s1,
Table S1: Proximal composition and energy value of the experimental breads: BYB, leavened with baker’s yeast
1.5% for 90 min at 30C; SB, sourdough bread leavened with S 4(20%) and baker’s yeast 1.5% for 90 min at 30C;
t-SB, sourdough bread leavened with S 24(20%) for 4 h at 30C. Table S2: Gastrointestinal motility: data resulting
from the study of the gastric and gallbladder emptying and H2 breath technique. BYB, leavened with baker’s
yeast 1.5% for 90 min at 30C; SB, sourdough bread leavened with S 4(20%) and baker’s yeast 1.5% for 90 min
at 30C; t-SB, sourdough bread leavened with S 24(20%) for 4 h at 30C. NU, Nutridrink, Figure S1: Flowchart
of breadmaking.
Author Contributions: C.G.R. was the supervisor of the research units, responsible of the experimental design and
of the data elaboration; L.B. and P .P . conceived the clinical experiments, analyzed the related data and contribute to
the writing of the article; M.M. carried out fermentations, bread making and microbial, chemical and technological
characterization of the samples; D.M.D.P . and M.P .L. collected samples and performed the clinical experiments;
B.G. provided food ingredients and was responsible for the funding; M.D.A. participated to the design of the
research and performed data evaluation; M.G. was the scientific advisor, conceived the work and wrote the article.
Funding: This research received no external funding.
Acknowledgments: The authors thank Puratos NV (Groot-Bijgaarden, Belgium) that promoted and sponsored
the research; Pietro and Andrea Minisci (Valle Fiorita Catering S.r.l., Ostuni, BR, Italy) for bread manufacturing;
Paola De Benedictis, Rosa De Venuto, Mariella Bruscella, Ornella de Bari for their skillful technical assistance.
Conflicts of Interest: The authors declare no competing financial and non-financial interests.
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