1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1Universidad Nacional Agraria La
Molina. Lima, Peru
2Associate Professor of Biology
Department.
*Corresponding
Carolina S. Huapaya Castillo
carolinahuapayacastillo@gmail.com
Received: 20 January 2022
Accepted: 05 May 2023
Published: 18 July 2023
Preparation of a probiotic quinoa beverage
by enzymatic hydrolysis of its starches and
subsequent lactic acid fermentation
Elaboración de una bebida probiótica de quinua por
hidrólisis enzimática de sus almidones y posterior
fermentación ácido láctica
Carolina S. Huapaya Castillo1 , Juan G. Juscamaita Morales1, 2
ABSTRACT
Background: The concern about consuming healthy foods has increased in recent years.
Not only are they expected to comply with essential feeding functions, but they also provide
health benefits. Probiotics are one of the main functional components expected to be
present in functional foods and beverages. They provide many health benefits and stand out
due to their metabolic capacities and adaptability to different habitats. In addition, Quinoa
seeds contain valuable quantities of quality protein and nutritional values of carbohydrates,
proteins, fats, fibers, and mineral substances for which they are considered an ideal dietary
alternative. Objectives: This research aimed to elaborate on a probiotic quinoa beverage,
which combines the effect of enzymatic hydrolysis of the starches obtained from its seeds
with lactic acid fermentation using probiotic cultures, seeking to enhance its nutritional
properties and converting it into a functional beverage. Methods: For this, fermentations
were carried out in three different concentrations of probiotic cultures (inoculum): 10%,
5%, 1%, and three other different fermentation times: 8, 10, and 12 hours. pH, Total titrable
acidity expressed as lactic acid (%), reducing sugars, and soluble solids were measured. After
that, the beverage was formulated with honey, carob, preservatives, and mango flavoring.
Results: Statistical analysis indicated optimal conditions were achieved with 10% probiotic
cultures and 10 hours of fermentation. The microbiological analysis confirmed the presence
of probiotic microorganisms at a concentration of 10 8 CFU/mL. Proximal analysis indicated
that the composition contained 84.6 Kcal, 19.3 g of carbohydrates, and 1.4 g of protein per
100 g of beverage. Conclusions: The probiotic quinoa beverage was produced and can be
considered in the group of plant-based foods, as well as a functional beverage, since the
probiotic cultures it contains contribute to maintaining the intestinal microbiota and prevent
the onset of chronic diseases.
Keywords: Probiotic quinoa beverage, Fermented functional beverages, Probiotics, Plant-
based foods.
ORIGINAL ARTICLE
Published 18 July 2023
Doi: https://doi.org/10.17533/udea.vitae.v30n2a352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1Universidad Nacional Agraria La
Molina. Lima, Peru
2Associate Professor of Biology
Department.
*Corresponding
Carolina S. Huapaya Castillo
carolinahuapayacastillo@gmail.com
Received: 20 January 2022
Accepted: 05 May 2023
Published: 18 July 2023
Preparation of a probiotic quinoa beverage
by enzymatic hydrolysis of its starches and
subsequent lactic acid fermentation
Elaboración de una bebida probiótica de quinua por
hidrólisis enzimática de sus almidones y posterior
fermentación ácido láctica
Carolina S. Huapaya Castillo1 , Juan G. Juscamaita Morales1, 2
ABSTRACT
Background: The concern about consuming healthy foods has increased in recent years.
Not only are they expected to comply with essential feeding functions, but they also provide
health benefits. Probiotics are one of the main functional components expected to be
present in functional foods and beverages. They provide many health benefits and stand out
due to their metabolic capacities and adaptability to different habitats. In addition, Quinoa
seeds contain valuable quantities of quality protein and nutritional values of carbohydrates,
proteins, fats, fibers, and mineral substances for which they are considered an ideal dietary
alternative. Objectives: This research aimed to elaborate on a probiotic quinoa beverage,
which combines the effect of enzymatic hydrolysis of the starches obtained from its seeds
with lactic acid fermentation using probiotic cultures, seeking to enhance its nutritional
properties and converting it into a functional beverage. Methods: For this, fermentations
were carried out in three different concentrations of probiotic cultures (inoculum): 10%,
5%, 1%, and three other different fermentation times: 8, 10, and 12 hours. pH, Total titrable
acidity expressed as lactic acid (%), reducing sugars, and soluble solids were measured. After
that, the beverage was formulated with honey, carob, preservatives, and mango flavoring.
Results: Statistical analysis indicated optimal conditions were achieved with 10% probiotic
cultures and 10 hours of fermentation. The microbiological analysis confirmed the presence
of probiotic microorganisms at a concentration of 10 8 CFU/mL. Proximal analysis indicated
that the composition contained 84.6 Kcal, 19.3 g of carbohydrates, and 1.4 g of protein per
100 g of beverage. Conclusions: The probiotic quinoa beverage was produced and can be
considered in the group of plant-based foods, as well as a functional beverage, since the
probiotic cultures it contains contribute to maintaining the intestinal microbiota and prevent
the onset of chronic diseases.
Keywords: Probiotic quinoa beverage, Fermented functional beverages, Probiotics, Plant-
based foods.
ORIGINAL ARTICLE
Published 18 July 2023
Doi: https://doi.org/10.17533/udea.vitae.v30n2a352397
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 352397Carolina S. Huapaya Castillo, Juan G. Juscamaita Morales
INTRODUCTION:
The functioning of the human body depends on
a complex interaction with the native microbiota
(1), which are microorganisms that establish a
symbiotic relationship with the host and helps
maintain physiological homeostasis (2). Native
microbiota is different for each individual, and its
composition depends on lifestyle, early exposure
to microorganisms in their environment, therapy
against infections (antibiotics), and the genotype
of the individual (1). The individual’s diet (3) is a
determining factor too.
Nevertheless, the current accelerated lifestyle,
the unhealthy eating habits (4), together with
factors such as a sedentary lifestyle and stress,
are influencing this microbiota (5), stimulating the
increase in diseases such as diabetes, obesity, high
blood pressure, and cancer (4). For example, a
diet predominating animal proteins and fats would
be associated with a higher incidence of obesity
(3), diabetes mellitus (6), metabolic syndrome,
inflammatory bowel disease, and coronary heart
disease. Many diseases are even the result of the
loss of harmony between our genome and the
microbiome (1). On the other hand, a diet with a
predominance of plant-based foods, with a good
proportion of prebiotics, would seek an intestinal
microbiota that is not associated with the mentioned
conditions and that could even reverse them. (3).
In this sense, consuming “plant-based foods” can
become a solution, as they can promote healthy
intestinal microbiota. Furthermore, they have
been promoted (7) since it was discovered that
they provide abundant functional phytochemicals,
such as carotenoids, polyphenols, flavonoids, and
saponins, which can reduce the risk of chronic
diseases (8). For these reasons, plant-based non-
alcoholic beverages have achieved a growth rate of
up to 15% in the global market in recent years (8).
For preparing these beverages, cereals are proposed
as optimal (9), considering they are an important
source of nutrients and functional phytochemicals (8).
On this basis, quinoa (Chenopodium quinoa Willd.)
is one of the most important. It is a dicotyledonous
plant belonging to the Chenopodiaceae family (10),
considered a pseudocereal native to the Andean
region. It contains minerals, proteins, and important
phytochemicals such as phenols, isoflavones,
saponins, and phytosterols (11). Without having an
exceptional amount of protein (from 12 to 23%), it
is characterized by its high combination of essential
amino acids because it has an ideal balance.
Regrettably, consumers do not entirely accept
cereals due to their sensory qualities and because
some of their nutrients’ digestibility is inadequate
for specific individuals (11). Therefore, they are
usually supplemented with enzymatic treatments
and microbial fermentations to improve their flavor
and nutrient transfer (11). In recent years, interest has
focused on obtaining fermented beverages based
on cereals or pseudocereals, which can be used as
substitutes for fermented beverages (12).
RESUMEN
Antecedentes: La preocupación por el consumo alimentos saludables ha aumentado en los últimos años. No solo se espera
que cumplan con las funciones esenciales de alimentación, sino que también brinden beneficios para la salud. Los probióticos
son uno de los principales componentes funcionales que se espera que estén presentes en los alimentos y bebidas funcionales.
Aportan múltiples beneficios para la salud y destacan por sus capacidades metabólicas y adaptabilidad a diferentes hábitats.
Además, las semillas de quinua contienen valiosas cantidades de proteína de notable calidad, valores nutricionales de
carbohidratos, proteínas, grasas, fibras y sustancias minerales por lo que se consideran una alternativa dietética ideal. Objetivos:
Esta investigación tuvo como objetivo elaborar una bebida probiótica de quinua, que combina el efecto de la hidrólisis
enzimática de los almidones obtenidos de sus semillas con la fermentación láctica utilizando cultivos probióticos, buscando
potenciar sus propiedades nutricionales y convertirla en una bebida funcional. Métodos: Para ello se realizaron fermentaciones
en tres concentraciones diferentes de cultivos probióticos (inóculo): 10%, 5%, 1%, y tres distintos tiempos de fermentación:
8, 10 y 12 horas. Se midió pH, Acidez titulable total expresada como ácido láctico (%), azúcares reductores y sólidos solubles.
Posteriormente, se formuló la bebida con miel, algarrobina, conservantes y saborizante de mango. Resultados: El análisis
estadístico indicó que se lograron condiciones óptimas con 10% de cultivos probióticos y 10 horas de fermentación. El análisis
microbiológico confirmó la presencia de microorganismos probióticos a una concentración de 10 8 UFC/mL. El análisis proximal
indicó que la composición contenía 84,6 Kcal, 19,3 g de carbohidratos y 1,4 g de proteína por 100 g de bebida. Conclusiones:
la bebida probiótica de quinua fue elaborada y puede ser considerada en el grupo de alimentos de origen vegetal, así como
una bebida funcional, ya que los cultivos probióticos que contiene contribuyen al mantenimiento de la microbiota intestinal y
previenen la aparición de enfermedades crónicas.
Palabras clave: Bebida probiótica de quinua, Bebidas funcionales fermentadas, probióticos, alimentos basados en plantas
INTRODUCTION:
The functioning of the human body depends on
a complex interaction with the native microbiota
(1), which are microorganisms that establish a
symbiotic relationship with the host and helps
maintain physiological homeostasis (2). Native
microbiota is different for each individual, and its
composition depends on lifestyle, early exposure
to microorganisms in their environment, therapy
against infections (antibiotics), and the genotype
of the individual (1). The individual’s diet (3) is a
determining factor too.
Nevertheless, the current accelerated lifestyle,
the unhealthy eating habits (4), together with
factors such as a sedentary lifestyle and stress,
are influencing this microbiota (5), stimulating the
increase in diseases such as diabetes, obesity, high
blood pressure, and cancer (4). For example, a
diet predominating animal proteins and fats would
be associated with a higher incidence of obesity
(3), diabetes mellitus (6), metabolic syndrome,
inflammatory bowel disease, and coronary heart
disease. Many diseases are even the result of the
loss of harmony between our genome and the
microbiome (1). On the other hand, a diet with a
predominance of plant-based foods, with a good
proportion of prebiotics, would seek an intestinal
microbiota that is not associated with the mentioned
conditions and that could even reverse them. (3).
In this sense, consuming “plant-based foods” can
become a solution, as they can promote healthy
intestinal microbiota. Furthermore, they have
been promoted (7) since it was discovered that
they provide abundant functional phytochemicals,
such as carotenoids, polyphenols, flavonoids, and
saponins, which can reduce the risk of chronic
diseases (8). For these reasons, plant-based non-
alcoholic beverages have achieved a growth rate of
up to 15% in the global market in recent years (8).
For preparing these beverages, cereals are proposed
as optimal (9), considering they are an important
source of nutrients and functional phytochemicals (8).
On this basis, quinoa (Chenopodium quinoa Willd.)
is one of the most important. It is a dicotyledonous
plant belonging to the Chenopodiaceae family (10),
considered a pseudocereal native to the Andean
region. It contains minerals, proteins, and important
phytochemicals such as phenols, isoflavones,
saponins, and phytosterols (11). Without having an
exceptional amount of protein (from 12 to 23%), it
is characterized by its high combination of essential
amino acids because it has an ideal balance.
Regrettably, consumers do not entirely accept
cereals due to their sensory qualities and because
some of their nutrients’ digestibility is inadequate
for specific individuals (11). Therefore, they are
usually supplemented with enzymatic treatments
and microbial fermentations to improve their flavor
and nutrient transfer (11). In recent years, interest has
focused on obtaining fermented beverages based
on cereals or pseudocereals, which can be used as
substitutes for fermented beverages (12).
RESUMEN
Antecedentes: La preocupación por el consumo alimentos saludables ha aumentado en los últimos años. No solo se espera
que cumplan con las funciones esenciales de alimentación, sino que también brinden beneficios para la salud. Los probióticos
son uno de los principales componentes funcionales que se espera que estén presentes en los alimentos y bebidas funcionales.
Aportan múltiples beneficios para la salud y destacan por sus capacidades metabólicas y adaptabilidad a diferentes hábitats.
Además, las semillas de quinua contienen valiosas cantidades de proteína de notable calidad, valores nutricionales de
carbohidratos, proteínas, grasas, fibras y sustancias minerales por lo que se consideran una alternativa dietética ideal. Objetivos:
Esta investigación tuvo como objetivo elaborar una bebida probiótica de quinua, que combina el efecto de la hidrólisis
enzimática de los almidones obtenidos de sus semillas con la fermentación láctica utilizando cultivos probióticos, buscando
potenciar sus propiedades nutricionales y convertirla en una bebida funcional. Métodos: Para ello se realizaron fermentaciones
en tres concentraciones diferentes de cultivos probióticos (inóculo): 10%, 5%, 1%, y tres distintos tiempos de fermentación:
8, 10 y 12 horas. Se midió pH, Acidez titulable total expresada como ácido láctico (%), azúcares reductores y sólidos solubles.
Posteriormente, se formuló la bebida con miel, algarrobina, conservantes y saborizante de mango. Resultados: El análisis
estadístico indicó que se lograron condiciones óptimas con 10% de cultivos probióticos y 10 horas de fermentación. El análisis
microbiológico confirmó la presencia de microorganismos probióticos a una concentración de 10 8 UFC/mL. El análisis proximal
indicó que la composición contenía 84,6 Kcal, 19,3 g de carbohidratos y 1,4 g de proteína por 100 g de bebida. Conclusiones:
la bebida probiótica de quinua fue elaborada y puede ser considerada en el grupo de alimentos de origen vegetal, así como
una bebida funcional, ya que los cultivos probióticos que contiene contribuyen al mantenimiento de la microbiota intestinal y
previenen la aparición de enfermedades crónicas.
Palabras clave: Bebida probiótica de quinua, Bebidas funcionales fermentadas, probióticos, alimentos basados en plantas
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
From this perspective, it is possible to formulate
products with probiotics (13) and food substances
needed to grow lactic acid bacteria (14). Probiotics
constitute one of the main functional components
and represent an accessible option to the population
(15). Lactic acid fermentation produces them
with functional ingredients such as short-chain
fatty acids, vitamins, and exopolysaccharides
(8). Likewise, they play a vital role in maintaining
intestinal microecology and preventing chronic
diseases (8). Lactic acid fermentation products can
even change the taste of food or beverages (11).
For the reasons mentioned above, this research
aims to develop a probiotic quinoa beverage
that combines plant-based foods with lactic acid
fermentation. Therefore, sensory qualities can be
improved, and the intestinal microbiota can be
positively impacted. Furthermore, the novelty lies
in using an enzymatic pretreatment to hydrolyze the
starch, considering that few reports use enzymatic
hydrolysis (11), and, on the other hand, fermentation
can enhance antioxidant activity (16).
MATERIALS AND METHODS
Starch hydrolysis of quinoa flour
Quinoa flour was purchased from local markets in
Lima, Perú, in the “Nutrimix” brand. It consisted
of crushed and ground white quinoa grains. Its
composition contains easily digestible proteins,
calcium, iron, phosphorus, and lysine (17). Following
the procedure described by (18), a suspension of
white quinoa flour (“Nutrimix” brand) was made at
12.5 % w/w. Subsequently, the pH was conditioned
to 7.8, and the enzyme α-amylase from Bacillus
licheniformis (Sigma Aldrich ® ) was added at a
concentration of 0.01% p/p starch, considering a
concentration of 70% starch. Next, it was heated at
100°C for 40 minutes in a water bath (Memmert) with
constant stirring. Later, the temperature was reduced
to 90 °C, and the hydrolysis was continued for another
60 minutes with continuous stirring. Finally, hydrolysis
was terminated by lowering the temperature to 25°C.
This stage released the sugars in the quinoa starch to
be lactically fermented in the next step. To confirm
the occurrence of hydrolysis, soluble solids (°Brix)
were measured before and after hydrolysis.
Lactic acid fermentation
A “Vivolac” brand probiotic culture suspension
was made at 0.278% w/w. The culture included
the genera: Lactobacillus delbrueckii subsp.
Bulgaricus (10%), Streptococcus salivarius subsp.
Thermophiles (70%), Lactobacillus acidophilus (10%)
and Bifidobacterium ssp. (10%) (19). This suspension
was added to the hydrolyzed starch solution in three
different concentrations: 10 , 5 , 1 , and 0 %, and the
suspension was carried to lactic acid fermentation
at 42 ± 1°C, under microaerophilic conditions in an
incubator (Memmert, Model 854 Schwbach). Three
different fermentation times were tested: 8, 10,
and 12 hours, obtaining 12 treatments performed
in triplicate. These times were determined based
on previous tests that stated that pH begins to
decrease after 6 hours. Thus, considering that these
cultures are for milk (19) and not for quinoa, it was
necessary to consider the adaptation time for the
microorganisms (20). Finally, the fermentations were
stopped by reducing the temperature to 4°C.
For all treatments, four parameters were measured:
pH (HANNA portable pH meter, Model HI 8424),
total titrable acidity expressed as a percentage
of lactic acid (% w/w) (according to the procedure
described by (21); reducing sugars by the DNS
method, described by Miller, and modified by
(22); and soluble solids (°Brix) (with an RHB-55
refractometer). These parameters were used as
indicators of the occurrence of fermentation and
were measured both before and after fermentation
had started.
For total acidity, the following formula was used:
0.1 *0.009*100
% mL NaOH M
Lactic acid mL of sample
=
Likewise, a calibration curve was prepared to
determine total sugars, and the spectrophotometer
readings at 540 nm were transformed into reducing
sugar concentration (mg/mL) using the equation y
= 6.579 X - 0.2414.
Once all the fermentations were finished, the best
treatment was chosen based on its lactic acid
production, according to the recommendations of
(23). Other parameters: pH, reducing sugars, and
soluble solids, were also measured. Still, they were
only considered as a reference).
Formulation
The beverage was formulated by adding natural
honey (4.375 % w/v), carob (2.5 % w/v), and
potassium sorbate (0.01 % w/v). Additionally, the
beverage was formulated by adding mango flavor
(with cloudy mango flavoring (7 drops/400 mL) from
Fratello Importaciones Goicochea EIRL).
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
From this perspective, it is possible to formulate
products with probiotics (13) and food substances
needed to grow lactic acid bacteria (14). Probiotics
constitute one of the main functional components
and represent an accessible option to the population
(15). Lactic acid fermentation produces them
with functional ingredients such as short-chain
fatty acids, vitamins, and exopolysaccharides
(8). Likewise, they play a vital role in maintaining
intestinal microecology and preventing chronic
diseases (8). Lactic acid fermentation products can
even change the taste of food or beverages (11).
For the reasons mentioned above, this research
aims to develop a probiotic quinoa beverage
that combines plant-based foods with lactic acid
fermentation. Therefore, sensory qualities can be
improved, and the intestinal microbiota can be
positively impacted. Furthermore, the novelty lies
in using an enzymatic pretreatment to hydrolyze the
starch, considering that few reports use enzymatic
hydrolysis (11), and, on the other hand, fermentation
can enhance antioxidant activity (16).
MATERIALS AND METHODS
Starch hydrolysis of quinoa flour
Quinoa flour was purchased from local markets in
Lima, Perú, in the “Nutrimix” brand. It consisted
of crushed and ground white quinoa grains. Its
composition contains easily digestible proteins,
calcium, iron, phosphorus, and lysine (17). Following
the procedure described by (18), a suspension of
white quinoa flour (“Nutrimix” brand) was made at
12.5 % w/w. Subsequently, the pH was conditioned
to 7.8, and the enzyme α-amylase from Bacillus
licheniformis (Sigma Aldrich ® ) was added at a
concentration of 0.01% p/p starch, considering a
concentration of 70% starch. Next, it was heated at
100°C for 40 minutes in a water bath (Memmert) with
constant stirring. Later, the temperature was reduced
to 90 °C, and the hydrolysis was continued for another
60 minutes with continuous stirring. Finally, hydrolysis
was terminated by lowering the temperature to 25°C.
This stage released the sugars in the quinoa starch to
be lactically fermented in the next step. To confirm
the occurrence of hydrolysis, soluble solids (°Brix)
were measured before and after hydrolysis.
Lactic acid fermentation
A “Vivolac” brand probiotic culture suspension
was made at 0.278% w/w. The culture included
the genera: Lactobacillus delbrueckii subsp.
Bulgaricus (10%), Streptococcus salivarius subsp.
Thermophiles (70%), Lactobacillus acidophilus (10%)
and Bifidobacterium ssp. (10%) (19). This suspension
was added to the hydrolyzed starch solution in three
different concentrations: 10 , 5 , 1 , and 0 %, and the
suspension was carried to lactic acid fermentation
at 42 ± 1°C, under microaerophilic conditions in an
incubator (Memmert, Model 854 Schwbach). Three
different fermentation times were tested: 8, 10,
and 12 hours, obtaining 12 treatments performed
in triplicate. These times were determined based
on previous tests that stated that pH begins to
decrease after 6 hours. Thus, considering that these
cultures are for milk (19) and not for quinoa, it was
necessary to consider the adaptation time for the
microorganisms (20). Finally, the fermentations were
stopped by reducing the temperature to 4°C.
For all treatments, four parameters were measured:
pH (HANNA portable pH meter, Model HI 8424),
total titrable acidity expressed as a percentage
of lactic acid (% w/w) (according to the procedure
described by (21); reducing sugars by the DNS
method, described by Miller, and modified by
(22); and soluble solids (°Brix) (with an RHB-55
refractometer). These parameters were used as
indicators of the occurrence of fermentation and
were measured both before and after fermentation
had started.
For total acidity, the following formula was used:
0.1 *0.009*100
% mL NaOH M
Lactic acid mL of sample
=
Likewise, a calibration curve was prepared to
determine total sugars, and the spectrophotometer
readings at 540 nm were transformed into reducing
sugar concentration (mg/mL) using the equation y
= 6.579 X - 0.2414.
Once all the fermentations were finished, the best
treatment was chosen based on its lactic acid
production, according to the recommendations of
(23). Other parameters: pH, reducing sugars, and
soluble solids, were also measured. Still, they were
only considered as a reference).
Formulation
The beverage was formulated by adding natural
honey (4.375 % w/v), carob (2.5 % w/v), and
potassium sorbate (0.01 % w/v). Additionally, the
beverage was formulated by adding mango flavor
(with cloudy mango flavoring (7 drops/400 mL) from
Fratello Importaciones Goicochea EIRL).
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 352397Carolina S. Huapaya Castillo, Juan G. Juscamaita Morales
Proximal analysis
The beverage was subjected to a proximal analysis in
“La Molina Calidad Total” laboratories. The following
parameters were analyzed: Total calories (by MS-IN
calculation, Collazos 1993); Carbohydrates (by MS-
INN difference, Collazos 1993); Fat (AOAC 986.35
(B). Cap 50, Ed.18, Page 18, Revision 4. 2011-2005);
Humidity (AOAC 950.27 (B). Chap 29, Ed.18, Page
6, Revision 4. 2011-2005); Ashes (AOAC 950.27 (B).
Cap 29, Ed.18, Page 2, Revision 4. 2011-2005); Protein
(AOAC 986.13 Chap 32, Ed.18, Page 14, Revision 4.
2011-2005); Crude fiber (NTP.205.003, Revised 2011.
1980); %Kcal from fat (By MS-INN calculation. Collazos
1993); % Kcal from protein (By MS-INN calculation.
Collazos 1993); % Kcal from carbohydrates (By MS-
INN calculation. Collazos 1993).
Microbiological analysis
The beverage was also microbiologically analyzed
two times: immediately after its preparation and two
weeks after it. During these two weeks, the product
was stored under aseptic conditions at a temperature
of 4°C. The parameters analyzed, as requested by
the General Directorate of Environmental Health
of the Republic of Peru (24) were: Total mesophilic
aerobic count by analysis in Plate Count Agar;
Count of fungi and yeasts by Analysis in Sabouraud
Agar; Total coliform count by analysis in VRB Agar;
Lactobacillus count by analysis in MRS Agar. In
addition, a simple gram staining of the colonies that
grew on MRS agar was also performed.
Statistical analysis
To determine the treatment with the highest
production of lactic acid, a 4x3 factorial experiment
was used, with three repetitions, suitable for a
DCA. Then, the analysis of variance (ANOVA)
conducted in DCA was performed, and a mean
comparison test (Tukey’s test) was applied for the
main and simple effects.
RESULTS
Enzymatic hydrolysis
After enzymatic hydrolysis, the measurement of
soluble solids (°Brix) indicated that, on average,
7.5 g of soluble solids were released per 100 g
of beverage, resulting in between 9 and 10 °Brix
at the end of hydrolysis. These results are similar
to those reported by (18), who, after following a
similar procedure, obtained a final 9 °Brix. Due to
the α-amylase from Bacillus licheniformis (Sigma-
Aldrich ® ), these released soluble solids would
consist of maltose, maltotriose, maltotetraoses,
maltooligosaccharides, and glucose (25), which
are susceptible to being fermented by probiotic
microorganisms in the next stage. It is worth
mentioning that previous tests showed that not
adding enzymes led to starch gelatinization.
Lactic acid fermentation
pH
For all treatments, after the fermentation, it was
observed a decrease in pH (Table 1), including the
control groups, although, in these, the variation was
minimal. The greatest pH variations were observed
in the 10-hour treatment for 10 % and 5 % inoculum.
A decrease in pH below 4.0 would be an indicator
of acid production. According to (26), determining
pH is essential in monitoring acid production by
lactic acid bacteria.
Table 1. pH (± 0.01) at different fermentation times and different inoculum concentrations
pH (± 0.01)
8 HOURS 10 HOURS 12 HOURS
INOCULUM INITIAL FINAL INITIAL FINAL INITIAL FINAL
0% 6.68a ± 0.02 6.57a ± 0.04 6.85e ± 0.05 6.14e ± 0.91 6.80j ± 0.06 6.29j ± 0.79
1% 6.66a ± 0.02 4.29b ± 0.01 6.83e ± 0.03 4.18f ± 0.04 6.74j ± 0.00 3.96k
± 0.05
5% 6.65a ± 0.01 4.14c ± 0.04 6.81e ± 0.05 3.86e ± 0.10 6.74j ± 0.03 3.90m ± 0.07
10% 6.57a ± 0.02 4.02d ± 0.03 6.79e ± 0.05 3.84h ± 0.09 6.64j ± 0.04 3.88n ± 0.05
* The same letter indicates that there were no significant differences between initial and final stages.
Proximal analysis
The beverage was subjected to a proximal analysis in
“La Molina Calidad Total” laboratories. The following
parameters were analyzed: Total calories (by MS-IN
calculation, Collazos 1993); Carbohydrates (by MS-
INN difference, Collazos 1993); Fat (AOAC 986.35
(B). Cap 50, Ed.18, Page 18, Revision 4. 2011-2005);
Humidity (AOAC 950.27 (B). Chap 29, Ed.18, Page
6, Revision 4. 2011-2005); Ashes (AOAC 950.27 (B).
Cap 29, Ed.18, Page 2, Revision 4. 2011-2005); Protein
(AOAC 986.13 Chap 32, Ed.18, Page 14, Revision 4.
2011-2005); Crude fiber (NTP.205.003, Revised 2011.
1980); %Kcal from fat (By MS-INN calculation. Collazos
1993); % Kcal from protein (By MS-INN calculation.
Collazos 1993); % Kcal from carbohydrates (By MS-
INN calculation. Collazos 1993).
Microbiological analysis
The beverage was also microbiologically analyzed
two times: immediately after its preparation and two
weeks after it. During these two weeks, the product
was stored under aseptic conditions at a temperature
of 4°C. The parameters analyzed, as requested by
the General Directorate of Environmental Health
of the Republic of Peru (24) were: Total mesophilic
aerobic count by analysis in Plate Count Agar;
Count of fungi and yeasts by Analysis in Sabouraud
Agar; Total coliform count by analysis in VRB Agar;
Lactobacillus count by analysis in MRS Agar. In
addition, a simple gram staining of the colonies that
grew on MRS agar was also performed.
Statistical analysis
To determine the treatment with the highest
production of lactic acid, a 4x3 factorial experiment
was used, with three repetitions, suitable for a
DCA. Then, the analysis of variance (ANOVA)
conducted in DCA was performed, and a mean
comparison test (Tukey’s test) was applied for the
main and simple effects.
RESULTS
Enzymatic hydrolysis
After enzymatic hydrolysis, the measurement of
soluble solids (°Brix) indicated that, on average,
7.5 g of soluble solids were released per 100 g
of beverage, resulting in between 9 and 10 °Brix
at the end of hydrolysis. These results are similar
to those reported by (18), who, after following a
similar procedure, obtained a final 9 °Brix. Due to
the α-amylase from Bacillus licheniformis (Sigma-
Aldrich ® ), these released soluble solids would
consist of maltose, maltotriose, maltotetraoses,
maltooligosaccharides, and glucose (25), which
are susceptible to being fermented by probiotic
microorganisms in the next stage. It is worth
mentioning that previous tests showed that not
adding enzymes led to starch gelatinization.
Lactic acid fermentation
pH
For all treatments, after the fermentation, it was
observed a decrease in pH (Table 1), including the
control groups, although, in these, the variation was
minimal. The greatest pH variations were observed
in the 10-hour treatment for 10 % and 5 % inoculum.
A decrease in pH below 4.0 would be an indicator
of acid production. According to (26), determining
pH is essential in monitoring acid production by
lactic acid bacteria.
Table 1. pH (± 0.01) at different fermentation times and different inoculum concentrations
pH (± 0.01)
8 HOURS 10 HOURS 12 HOURS
INOCULUM INITIAL FINAL INITIAL FINAL INITIAL FINAL
0% 6.68a ± 0.02 6.57a ± 0.04 6.85e ± 0.05 6.14e ± 0.91 6.80j ± 0.06 6.29j ± 0.79
1% 6.66a ± 0.02 4.29b ± 0.01 6.83e ± 0.03 4.18f ± 0.04 6.74j ± 0.00 3.96k
± 0.05
5% 6.65a ± 0.01 4.14c ± 0.04 6.81e ± 0.05 3.86e ± 0.10 6.74j ± 0.03 3.90m ± 0.07
10% 6.57a ± 0.02 4.02d ± 0.03 6.79e ± 0.05 3.84h ± 0.09 6.64j ± 0.04 3.88n ± 0.05
* The same letter indicates that there were no significant differences between initial and final stages.
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
A T-test was applied to test the differences between
the Initial) and the Final pH of the fermentation at
8, 10, and 12 hours. Results indicated significant
differences for all treatments (α=0.01), except
for those with 0% of inoculum. The medium was
rapidly acidified with higher concentrations of
inoculum since a higher initial population would
allow for reaching the maximum population size in
a considerably shorter time (27). However, as the
fermentation time increases, the pH values tend
to become similar, which could be because the
stationary phase has been reached.
Total Titrable acidity expressed as a percentage
of lactic acid (%)
As shown in Figure 1, the highest acidity productions
occurred in the 10-hour treatment and 10 %
inoculum, and the 12-hour treatment for 5 % and
1 % inoculum. These results are similar to those
found for the measurement of pH. Likewise, as the
fermentation time increases (12 hours), the acidity
values tend to become similar, indicating that
the stationary phase is being reached. It was also
observed that the production of lactic acid at 12
hours with 10 % inoculum is reduced, compared to
the concentration with 5 %. According to (28), the
accumulation of lactic acid results in adverse effects,
such as intracellular acidification, accumulation of
anions, membrane disturbances, and increase in
turgor pressure, among others.
The pH and total titrable acidity results indicate
that the microorganisms have adapted and are
using the carbohydrates in the quinoa starch to
initiate fermentation (23). The acid produced would
be mainly lactic acid since the probiotic strains used
are mostly homofermentative, and they have almost
exclusively lactate through the Embden-Meyerhof-
Parnas pathway (26).
-0,1
-0,05
0
0,05
0,1
0,15
0,2
0,25
0,3
0% 1% 5% 10%
Total Titrable acidity
(% lactic acid w/w)
8 HOURS 10 HOURS 12 HOURS
Figure 1. Total titrable acidity produced at different fermentation times and different inoculum concentrations. Error bars represent
standard deviation (3 replicates).
Reducing sugars:
Table 2 shows the data on the reducing sugars
concentration for all treatments before and after
fermentation. In the treatments at 8 hours, for
1 %, 5 %, and 10 % inoculum, an increase in the
concentration of reducing sugars is observed.
However, in the treatments at 10 and 12 hours,
a slight reduction is observed for 1 %, 5 %, and
10 % inoculum.
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
A T-test was applied to test the differences between
the Initial) and the Final pH of the fermentation at
8, 10, and 12 hours. Results indicated significant
differences for all treatments (α=0.01), except
for those with 0% of inoculum. The medium was
rapidly acidified with higher concentrations of
inoculum since a higher initial population would
allow for reaching the maximum population size in
a considerably shorter time (27). However, as the
fermentation time increases, the pH values tend
to become similar, which could be because the
stationary phase has been reached.
Total Titrable acidity expressed as a percentage
of lactic acid (%)
As shown in Figure 1, the highest acidity productions
occurred in the 10-hour treatment and 10 %
inoculum, and the 12-hour treatment for 5 % and
1 % inoculum. These results are similar to those
found for the measurement of pH. Likewise, as the
fermentation time increases (12 hours), the acidity
values tend to become similar, indicating that
the stationary phase is being reached. It was also
observed that the production of lactic acid at 12
hours with 10 % inoculum is reduced, compared to
the concentration with 5 %. According to (28), the
accumulation of lactic acid results in adverse effects,
such as intracellular acidification, accumulation of
anions, membrane disturbances, and increase in
turgor pressure, among others.
The pH and total titrable acidity results indicate
that the microorganisms have adapted and are
using the carbohydrates in the quinoa starch to
initiate fermentation (23). The acid produced would
be mainly lactic acid since the probiotic strains used
are mostly homofermentative, and they have almost
exclusively lactate through the Embden-Meyerhof-
Parnas pathway (26).
-0,1
-0,05
0
0,05
0,1
0,15
0,2
0,25
0,3
0% 1% 5% 10%
Total Titrable acidity
(% lactic acid w/w)
8 HOURS 10 HOURS 12 HOURS
Figure 1. Total titrable acidity produced at different fermentation times and different inoculum concentrations. Error bars represent
standard deviation (3 replicates).
Reducing sugars:
Table 2 shows the data on the reducing sugars
concentration for all treatments before and after
fermentation. In the treatments at 8 hours, for
1 %, 5 %, and 10 % inoculum, an increase in the
concentration of reducing sugars is observed.
However, in the treatments at 10 and 12 hours,
a slight reduction is observed for 1 %, 5 %, and
10 % inoculum.
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 352397Carolina S. Huapaya Castillo, Juan G. Juscamaita Morales
A T-test was applied to test the differences
between the beginning (Initial) and the end (Final)
of the fermentation at 8, 10, and 12 hours. Results
indicated no significant differences (α=0.05), except
for the 12-hours fermentation and 0% of inoculum.
According to (12), in cooked quinoa, sugars and
starch rise to 17 g / 100 g; while in quinoa beverages,
the values reach 3.4 g / 100 g. According to (29),
these sugars consist of glucose 1.7 g, fructose 0.2
g, sucrose 2.9 g, and maltose 1.4 g (g / 100 g dry
matter). Therefore, once the free sugars in quinoa
and those released by enzymatic hydrolysis have
been consumed, the probiotic microorganisms
would begin to consume other carbon sources.
Furthermore, since glucose, fructose, and other
sugars are metabolized by lactic acid bacteria (30),
starch will likely hydrolyze and generate glucose and
fructose as products. Therefore, it can be assumed
Table 2. Reducing sugars (± 0.01) at different fermentation times and different inoculum concentrations
Reducing sugars (mg/mL)
8 HOURS 10 HOURS 12 HOURS
INOCULUM INITIAL FINAL INITIAL FINAL INITIAL FINAL
0% 14.75a ± 020 14.18 a ± 0.30 15.46 e ± 1.40 14.84 e ± 0.76 14.34j ± 0.61 15.45 k ± 0.30
1% 13.31b ± 0.73 13.63 b ± 0.52 15.07 f ± 1.44 14.97 f ± 0.85 14.65l ± 0.61 14.90l ± 0.16
5% 12.83c ± 0.18 13.20c ± 0.49 13.96g ± 0.51 13.10 g ± 0.42 13.90m ± 0.56 13.62 m ± 0.89
10% 11.78 d ± 1.53 12.10d ± 1.35 14.38h ± 0.35 14.20 h ± 1.09 13.03 n ± 0.35 12.99 n ± 0.46
* The same letter indicates that there were no significant differences between initial and final stages.
that microorganisms immediately consume the
sugars released by their enzymatic action, which
would explain why there is no significant variation
in reducing sugars.
Soluble solids:
Table 3 shows the variation of soluble solids for all
treatments. It was observed that the soluble solids
vary in different patterns. For example, in the 8 and
10-hour treatments, the soluble solids decrease
while they increase in the 12-hour treatments.
Some controls showed variations too. As stated by
(31), soluble solids represent an important answer
in the study of a fermentation process. These
parameter changes include the growth of lactic
acid bacteria and the production of organic acids
during fermentation.
Table 3. Soluble solids (°Brix ± 0.01) at different fermentation times and different inoculum concentrations
Soluble solids (°Brix ± 0.1)
8 HOURS 10 HOURS 12 HOURS
INOCULUM INITIAL FINAL INITIAL FINAL INITIAL FINAL
0% 10.00a ± 0.00 10.00 a ± 0.00 10.00 a ± 0.00 8.83 f ± 0.29 9.33 m ± 0.58 9.67 m ± 0.29
1% 10.00a ± 0.00 9.17b ± 0.29 9.67g ± 0.29 8.83 g ± 0.76 9.00n ± 0.00 9.50p ± 0.00
5% 9.00 c ± 0.00 8.50d ± 0.00 8.50h ± 0.87 7.50 h ± 0.50 8.17q ± 0.29 8.67 q ± 0.29
10% 9.00 c ± 0.00 8.50 e ± 0.00 9.17i ± 0.29 7.17 k ± 0.29 8.00 r ± 0.00 8.17 r ± 0.29
* The same letter indicates that there were no significant differences between initial and final stages.
The decreases in the 8 and 10-hour treatments
coincide with the decreases in pH, suggesting that
free sugars are being used to produce lactic acid. In
the case of the 12-hour treatments, an increase was
evident for all inoculum concentrations, including
those of the control treatment. This result was
similar to that obtained by Cañón Rodríguez (32),
who stated that soluble solids increased in the final
A T-test was applied to test the differences
between the beginning (Initial) and the end (Final)
of the fermentation at 8, 10, and 12 hours. Results
indicated no significant differences (α=0.05), except
for the 12-hours fermentation and 0% of inoculum.
According to (12), in cooked quinoa, sugars and
starch rise to 17 g / 100 g; while in quinoa beverages,
the values reach 3.4 g / 100 g. According to (29),
these sugars consist of glucose 1.7 g, fructose 0.2
g, sucrose 2.9 g, and maltose 1.4 g (g / 100 g dry
matter). Therefore, once the free sugars in quinoa
and those released by enzymatic hydrolysis have
been consumed, the probiotic microorganisms
would begin to consume other carbon sources.
Furthermore, since glucose, fructose, and other
sugars are metabolized by lactic acid bacteria (30),
starch will likely hydrolyze and generate glucose and
fructose as products. Therefore, it can be assumed
Table 2. Reducing sugars (± 0.01) at different fermentation times and different inoculum concentrations
Reducing sugars (mg/mL)
8 HOURS 10 HOURS 12 HOURS
INOCULUM INITIAL FINAL INITIAL FINAL INITIAL FINAL
0% 14.75a ± 020 14.18 a ± 0.30 15.46 e ± 1.40 14.84 e ± 0.76 14.34j ± 0.61 15.45 k ± 0.30
1% 13.31b ± 0.73 13.63 b ± 0.52 15.07 f ± 1.44 14.97 f ± 0.85 14.65l ± 0.61 14.90l ± 0.16
5% 12.83c ± 0.18 13.20c ± 0.49 13.96g ± 0.51 13.10 g ± 0.42 13.90m ± 0.56 13.62 m ± 0.89
10% 11.78 d ± 1.53 12.10d ± 1.35 14.38h ± 0.35 14.20 h ± 1.09 13.03 n ± 0.35 12.99 n ± 0.46
* The same letter indicates that there were no significant differences between initial and final stages.
that microorganisms immediately consume the
sugars released by their enzymatic action, which
would explain why there is no significant variation
in reducing sugars.
Soluble solids:
Table 3 shows the variation of soluble solids for all
treatments. It was observed that the soluble solids
vary in different patterns. For example, in the 8 and
10-hour treatments, the soluble solids decrease
while they increase in the 12-hour treatments.
Some controls showed variations too. As stated by
(31), soluble solids represent an important answer
in the study of a fermentation process. These
parameter changes include the growth of lactic
acid bacteria and the production of organic acids
during fermentation.
Table 3. Soluble solids (°Brix ± 0.01) at different fermentation times and different inoculum concentrations
Soluble solids (°Brix ± 0.1)
8 HOURS 10 HOURS 12 HOURS
INOCULUM INITIAL FINAL INITIAL FINAL INITIAL FINAL
0% 10.00a ± 0.00 10.00 a ± 0.00 10.00 a ± 0.00 8.83 f ± 0.29 9.33 m ± 0.58 9.67 m ± 0.29
1% 10.00a ± 0.00 9.17b ± 0.29 9.67g ± 0.29 8.83 g ± 0.76 9.00n ± 0.00 9.50p ± 0.00
5% 9.00 c ± 0.00 8.50d ± 0.00 8.50h ± 0.87 7.50 h ± 0.50 8.17q ± 0.29 8.67 q ± 0.29
10% 9.00 c ± 0.00 8.50 e ± 0.00 9.17i ± 0.29 7.17 k ± 0.29 8.00 r ± 0.00 8.17 r ± 0.29
* The same letter indicates that there were no significant differences between initial and final stages.
The decreases in the 8 and 10-hour treatments
coincide with the decreases in pH, suggesting that
free sugars are being used to produce lactic acid. In
the case of the 12-hour treatments, an increase was
evident for all inoculum concentrations, including
those of the control treatment. This result was
similar to that obtained by Cañón Rodríguez (32),
who stated that soluble solids increased in the final
7Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
fermentation moments between 3.5 and 4.5 hours.
Additionally, in research carried out by Lima et al.
(31) in which they used Lactobacillus gasseri to
produce probiotic food, soluble solids increased
from 8 to 12 hours and decreased after 16 hours of
fermentation.
Statistical analysis
The best treatment was determined based on the
“Total Titrable acidity produced parameter. For
this, a two-factor ANOVA was applied. For the
effect of fermentation time, Fcal 27.63392 > F.tab
3.4; for the factor Concentration of probiotic
microorganisms, F cal 119.3991 > F.tab 3.01;
and for factor interaction, F.cal 3.105352 > F.tab
2.51. In all cases, Fcal>Ftab; therefore, both the
fermentation time and the concentration of probiotic
microorganisms significantly affected the samples.
Afterward, a comparison test was made for the main
and simple effects. The results indicated significant
differences between the 8-hour and 10-hour
fermentation but no significant differences between
the 10-hour and 12-hour fermentation. Therefore,
the fermentation of 10 hours was determined as
the best treatment. The tests for simple effects
concluded that, for a 10-hour fermentation, there
were no significant differences between 10 % and 5
% of the concentration of probiotics, but there were
between 5 % and 1 %. Using the 10 % concentration
as a preventive measure was preferred to ensure
the product’s safety since an initial population
of 10 % has a greater probability of eliminating
possible pathogens. In addition, it can reduce
chemical preservative usage in food and can also
have the potential to work in synergy with synthetic
preservatives (33). Thus, together with potassium
sorbate, it could extend the life of the product.
Consequently, the best treatment was 10 hours of
fermentation at a probiotic concentration of 10 %.
Proximal analysis
The formulated beverage, in mango flavor, prepared
with 10 hours of fermentation and 10 % inoculum,
was analyzed in La Molina Total Quality Laboratories.
The probiotic quinoa beverage contains 84.6
kcal/100 g of sample for the caloric content. This
energy was attributed to carob and honey, which
provided sugars to the beverage. Regarding
protein, its value is 1.4 g/100 g of beverage. It was
understood that the value of the protein was about
the dilution made (approximately one-tenth of the
content in the quinoa grain).
Regarding carbohydrates, the probiotic quinoa
beverage contains 19.3 g per 100 g, representing
an advantage if it is intended to be consumed as
an energy beverage. As for fat, the probiotic quinoa
beverage contains 0.2 g/100 g. Crude fiber amounts
to 0.1 g/100 g of sample.
MICROBIOLOGICAL ANALYSIS
The results of the microbiological analysis are shown
in Table 5.
Table 5. Results of the microbiological analysis.
Microbiological analysis (CFU/mL)
At the end of fermentation Two weeks later
Total mesophilic aerobic bacteria 1.15 x 108 CFU/mL 7.95 x 106 CFU/mL
Total coliforms < 3 CFU/mL < 3 CFU/mL
Molds and yeasts < 3 CFU/mL < 3 CFU/mL
Lactobacillus spp. 6.25 x 10 7 CFU/mL 3.9 x 106 CFU/mL
Considering that the inoculum was 10 % (2.53
x 10 6 CFU/mL), it was verified that the probiotic
microorganisms have multiplied in the quinoa
beverage. Analysis after two weeks confirms that
the probiotic microorganisms were still present
in the beverage, although in smaller quantities.
This suggested that the beverage will continue to
have the desired effect, even after two weeks of
preparation. Gram stain results of a colony grown
on MRS agar (Figure 2) revealed the presence of
bacilli in chains (Streptobacilli).
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
fermentation moments between 3.5 and 4.5 hours.
Additionally, in research carried out by Lima et al.
(31) in which they used Lactobacillus gasseri to
produce probiotic food, soluble solids increased
from 8 to 12 hours and decreased after 16 hours of
fermentation.
Statistical analysis
The best treatment was determined based on the
“Total Titrable acidity produced parameter. For
this, a two-factor ANOVA was applied. For the
effect of fermentation time, Fcal 27.63392 > F.tab
3.4; for the factor Concentration of probiotic
microorganisms, F cal 119.3991 > F.tab 3.01;
and for factor interaction, F.cal 3.105352 > F.tab
2.51. In all cases, Fcal>Ftab; therefore, both the
fermentation time and the concentration of probiotic
microorganisms significantly affected the samples.
Afterward, a comparison test was made for the main
and simple effects. The results indicated significant
differences between the 8-hour and 10-hour
fermentation but no significant differences between
the 10-hour and 12-hour fermentation. Therefore,
the fermentation of 10 hours was determined as
the best treatment. The tests for simple effects
concluded that, for a 10-hour fermentation, there
were no significant differences between 10 % and 5
% of the concentration of probiotics, but there were
between 5 % and 1 %. Using the 10 % concentration
as a preventive measure was preferred to ensure
the product’s safety since an initial population
of 10 % has a greater probability of eliminating
possible pathogens. In addition, it can reduce
chemical preservative usage in food and can also
have the potential to work in synergy with synthetic
preservatives (33). Thus, together with potassium
sorbate, it could extend the life of the product.
Consequently, the best treatment was 10 hours of
fermentation at a probiotic concentration of 10 %.
Proximal analysis
The formulated beverage, in mango flavor, prepared
with 10 hours of fermentation and 10 % inoculum,
was analyzed in La Molina Total Quality Laboratories.
The probiotic quinoa beverage contains 84.6
kcal/100 g of sample for the caloric content. This
energy was attributed to carob and honey, which
provided sugars to the beverage. Regarding
protein, its value is 1.4 g/100 g of beverage. It was
understood that the value of the protein was about
the dilution made (approximately one-tenth of the
content in the quinoa grain).
Regarding carbohydrates, the probiotic quinoa
beverage contains 19.3 g per 100 g, representing
an advantage if it is intended to be consumed as
an energy beverage. As for fat, the probiotic quinoa
beverage contains 0.2 g/100 g. Crude fiber amounts
to 0.1 g/100 g of sample.
MICROBIOLOGICAL ANALYSIS
The results of the microbiological analysis are shown
in Table 5.
Table 5. Results of the microbiological analysis.
Microbiological analysis (CFU/mL)
At the end of fermentation Two weeks later
Total mesophilic aerobic bacteria 1.15 x 108 CFU/mL 7.95 x 106 CFU/mL
Total coliforms < 3 CFU/mL < 3 CFU/mL
Molds and yeasts < 3 CFU/mL < 3 CFU/mL
Lactobacillus spp. 6.25 x 10 7 CFU/mL 3.9 x 106 CFU/mL
Considering that the inoculum was 10 % (2.53
x 10 6 CFU/mL), it was verified that the probiotic
microorganisms have multiplied in the quinoa
beverage. Analysis after two weeks confirms that
the probiotic microorganisms were still present
in the beverage, although in smaller quantities.
This suggested that the beverage will continue to
have the desired effect, even after two weeks of
preparation. Gram stain results of a colony grown
on MRS agar (Figure 2) revealed the presence of
bacilli in chains (Streptobacilli).
8Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 352397Carolina S. Huapaya Castillo, Juan G. Juscamaita Morales
Figure 2. Colonies of Streptobacilli (1000X) grown on MRS agar.
At a magnification of 1000X, the image suggested
that it would be the species Lactobacillus delbrueckii
subsp. bulgaricus and Lactobacillus acidophilus,
as these genera are gram-positive rods. The
information provided by (19) stated that the
probiotic microorganisms used include the genera
mentioned above.
Discussion:
The beverage in this research contained between
0.10 and 0.23 % w/w of lactic acid. This concentration
was lower than that reported in the work of Díaz &
Villa Fonseca (34), who elaborated a yogurt from
yellow pitahaya, finding acidity values between
0.6 % w/w and 0.7 % p/p. Secondly, our results were
similar to those found by Correa & Cuenca (23),
who showed that around 24 hours of fermentation
in some soybean beverages were required to reach
adequate lactic acid levels (this is a pH between 4
and 5). This could be explained by considering that
milk and other fermented dairy products provide
probiotic matrices (35). On the other hand, quinoa
-although a rich source of nutrients- has a different
composition than milk. In this sense, organisms such
as Lactobacillus delbrueckii subsp. bulgaricus, which
not only ferments lactose, would be very suitable for
vegetable substrates; instead, L. delbrueckii subsp.
Lactis that have the enzyme β-galactosidase could
only react in the presence of lactose (23)
On the other hand, even when the percentage of
lactic acid was lower, the pH reached inferior values
than those found in yogurt. This result was similar
to Cuenca et al. (36); although the production of
lactic acid in a beverage fermented from soy was
less than that of a beverage fermented from milk
(yogurt), the pH reached the same values for both
beverages. The reason would be the amount of
protein in these foods since the abundant amino
acids in milk proteins can act as buffer systems that
regulate pH variations. Thus, with an increase in the
concentration of H+ protons, the amino group of
the protein accepts these protons (37).
The probiotic quinoa beverage provides more
calories than other dairy probiotics considering the
proximal analysis. It is 12 times more energetic than
VitaBiosa, and about 500 times more than BioLaive
yogurt and Kevita beverages (38). Regarding
proteins, our product contained more protein than
VitaBiosa and Kevita beverages but less protein
than BioLaive yogurt (38). It should be considered
that compared with cereal crops (rice, maize, and
wheat), quinoa includes most of the essential amino
acids, with a composition that is adequate to the
adults’ requirements (39). Regarding carbohydrates,
the probiotic quinoa beverage had a similar
concentration of carbohydrates to that offered by
some probiotic yogurts (BioLaive yogurt 15.5 g,
VitaBiosa 1.5 g, and Kevita 3 g (38). Compared to
other beverages, it had less fat than BioLaive yogurt,
but higher fat than VitaBiosa and Kevita beverages
(38). While BioLaive contains 2 g of fat per 100 mL
of beverage, VitaBiosa and Kevita contain no fat,
and the probiotic beverage contains 0.2 g. In this
sense, the probiotic quinoa beverage was low in
fat compared to existing beverages on the market.
Considering that traditional yogurts do not have
dietary fiber, using pseudocereals -instead of milk- to
make beverages would be an alternative to improve
the functionality of this type of food (23).
In a study by Arenas-Suescún et al. (40), lactic
fermentation was carried out from milk with quinoa.
They found a final count of 5.61x10 6 CFU /mL after
3.7 hours. The same author reports that the counts
required for a commercial yogurt are around
107 CFU/mL. Likewise, according to Shori et. al.
(41), to ensure the health benefits of plant-based
fermented dairy products, probiotics must meet
a minimum level of probiotic bacteria between
106 and 107 CFU/mL until the date of expiration. In
this research, the probiotic microorganisms in the
beverage are in the order of 10 8 at the end of its
preparation and in the order of 10 6 up to two weeks
after it. This ensures that, when consumed, it will
provide the expected beneficial effects on health.
Notably, lactic acid bacteria are beneficial because
they can prevent the growth of harmful enteric
pathogens, supply enzymes, eliminate toxic food
elements in the intestine, promote immunomodulatory
action, stimulate the immune system, and enhance
the peristaltic action of the gastrointestinal tract (42).
Figure 2. Colonies of Streptobacilli (1000X) grown on MRS agar.
At a magnification of 1000X, the image suggested
that it would be the species Lactobacillus delbrueckii
subsp. bulgaricus and Lactobacillus acidophilus,
as these genera are gram-positive rods. The
information provided by (19) stated that the
probiotic microorganisms used include the genera
mentioned above.
Discussion:
The beverage in this research contained between
0.10 and 0.23 % w/w of lactic acid. This concentration
was lower than that reported in the work of Díaz &
Villa Fonseca (34), who elaborated a yogurt from
yellow pitahaya, finding acidity values between
0.6 % w/w and 0.7 % p/p. Secondly, our results were
similar to those found by Correa & Cuenca (23),
who showed that around 24 hours of fermentation
in some soybean beverages were required to reach
adequate lactic acid levels (this is a pH between 4
and 5). This could be explained by considering that
milk and other fermented dairy products provide
probiotic matrices (35). On the other hand, quinoa
-although a rich source of nutrients- has a different
composition than milk. In this sense, organisms such
as Lactobacillus delbrueckii subsp. bulgaricus, which
not only ferments lactose, would be very suitable for
vegetable substrates; instead, L. delbrueckii subsp.
Lactis that have the enzyme β-galactosidase could
only react in the presence of lactose (23)
On the other hand, even when the percentage of
lactic acid was lower, the pH reached inferior values
than those found in yogurt. This result was similar
to Cuenca et al. (36); although the production of
lactic acid in a beverage fermented from soy was
less than that of a beverage fermented from milk
(yogurt), the pH reached the same values for both
beverages. The reason would be the amount of
protein in these foods since the abundant amino
acids in milk proteins can act as buffer systems that
regulate pH variations. Thus, with an increase in the
concentration of H+ protons, the amino group of
the protein accepts these protons (37).
The probiotic quinoa beverage provides more
calories than other dairy probiotics considering the
proximal analysis. It is 12 times more energetic than
VitaBiosa, and about 500 times more than BioLaive
yogurt and Kevita beverages (38). Regarding
proteins, our product contained more protein than
VitaBiosa and Kevita beverages but less protein
than BioLaive yogurt (38). It should be considered
that compared with cereal crops (rice, maize, and
wheat), quinoa includes most of the essential amino
acids, with a composition that is adequate to the
adults’ requirements (39). Regarding carbohydrates,
the probiotic quinoa beverage had a similar
concentration of carbohydrates to that offered by
some probiotic yogurts (BioLaive yogurt 15.5 g,
VitaBiosa 1.5 g, and Kevita 3 g (38). Compared to
other beverages, it had less fat than BioLaive yogurt,
but higher fat than VitaBiosa and Kevita beverages
(38). While BioLaive contains 2 g of fat per 100 mL
of beverage, VitaBiosa and Kevita contain no fat,
and the probiotic beverage contains 0.2 g. In this
sense, the probiotic quinoa beverage was low in
fat compared to existing beverages on the market.
Considering that traditional yogurts do not have
dietary fiber, using pseudocereals -instead of milk- to
make beverages would be an alternative to improve
the functionality of this type of food (23).
In a study by Arenas-Suescún et al. (40), lactic
fermentation was carried out from milk with quinoa.
They found a final count of 5.61x10 6 CFU /mL after
3.7 hours. The same author reports that the counts
required for a commercial yogurt are around
107 CFU/mL. Likewise, according to Shori et. al.
(41), to ensure the health benefits of plant-based
fermented dairy products, probiotics must meet
a minimum level of probiotic bacteria between
106 and 107 CFU/mL until the date of expiration. In
this research, the probiotic microorganisms in the
beverage are in the order of 10 8 at the end of its
preparation and in the order of 10 6 up to two weeks
after it. This ensures that, when consumed, it will
provide the expected beneficial effects on health.
Notably, lactic acid bacteria are beneficial because
they can prevent the growth of harmful enteric
pathogens, supply enzymes, eliminate toxic food
elements in the intestine, promote immunomodulatory
action, stimulate the immune system, and enhance
the peristaltic action of the gastrointestinal tract (42).
9Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
CONCLUSIONS
Enzymatic hydrolysis with α-amylase allowed
the hydrolysis of quinoa flour starch. Lactic acid
fermentation with 10 % inoculum and 10 hours
of fermentation was the most suitable treatment
to obtain a probiotic quinoa beverage. The
proximal analysis confirmed that the beverage has
84.6 kcal/100 g, 19.3 g carbohydrates/100 g,
0.2 g fat/100 g, and 1.4 g protein/100 g per sample.
Lactobacillus spp. is present in the beverage at
a concentration of 6.25 x 107 CFU/mL at the end
of fermentation and 3.9 x 10 6 CFU/mL two weeks
later. Therefore, the consumption of the probiotic
quinoa beverage, in the recommended doses,
would allow making the most of the nutritional
benefits of quinoa and the beneficial effects of the
probiotic microorganisms, being able to prevent the
appearance of chronic non-communicable diseases.
The benefits of consuming probiotic microorganisms
are, among many others: prevention of diarrhea,
reduction in symptoms associated with inflammatory
bowel disease, prevention of gastrointestinal
cancers, alleviation of lactose intolerance, reduction
in Helicobacter pylori infection, prevention of
intestinal inflammatory disorders and regulation
of metabolic disorders, such as serum lipids,
cholesterol levels, blood pressure and glucose
regulation (43).
It is recommended for future research to determine
the percentage of survival of probiotic cultures
over a more extended period, simulating shelf life.
Likewise, it would be convenient to evaluate the
sensory acceptance and physicochemical stability
(pH, titratable acidity) after 21 days of storage at
different temperatures, as recommended by (44).
In addition, cofactors and prebiotics that could
enhance the yield of lactic acid would be a field to
explore since, in this investigation, values of only
0.2% w/w were reached as a maximum. Furthermore,
the determination of organic acids, free amino acids,
and antioxidant activity (45), the addition or absence
of preservatives, and the behavior of the beverage
without adding additives would complement the
information on this functional quinoa beverage.
Finally, the exploration of different genera of
lactic bacteria that could improve the beverage’s
quality would be important. Weissella confusa is
recommended by Boukid set al. (46) due to its
capacity to improve viscosity and mouthfeel in
fermented products.
CONFLICT OF INTEREST
None to declare
ACKNOWLEDGEMENT
We acknowledge the Environmental Biotechnology
- Bioremediation Laboratory (Department of
Biology, Universidad Nacional Agraria La Molina),
for providing us with the facilities for the research.
AUTHORS’ CONTRIBUTIONS
CSHC wrote the manuscript and provided data for
all tables. JGJM supervised the research. All authors
reviewed the final manuscript.
REFERENCES:
1. Álvarez-Calatayud, G., Guarner, F., Requena, T., & Marcos, A. (2018).
Dieta y microbiota. Impacto en la salud. Nutrición hospitalaria:
órgano oficial de la Sociedad Española de Nutrición Parenteral y
Enteral, 35(Spec6), 11–15. https://doi.org/10.20960/nh.2280
2. Requena, T., & Velasco, M. (2021). Microbioma humano en la
salud y la enfermedad. Revista Clínica Española, 221(4), 233–240.
https://doi.org/10.1016/j.rce.2019.07.004
3. Ursa Herguedas, A. J.; Ursa Bartolomé, M. I. (2022). Manejo de
la microbiota intestinal en la prevención y tratamiento de las
enfermedades de la civilización. Medicina Naturista. 16 (1), 9-13.
4. Maza-Avila, F. J., Caneda-Bermejo, M. C., & Vivas-Castillo, A.
C. (2022). Hábitos alimenticios y sus efectos en la salud de
los estudiantes universitarios. Una revisión sistemática de la
literatura. Psicogente, 25(47), 1–31. https://doi.org/10.17081/
psico.25.47.4861
5. Divella, R., De Palma, G., Tufaro, A., Pelagio, G., Gadaleta-
Caldarola, G., Bringiotti, R., & Paradiso, A. (2021). Diet, probiotics
and physical activity: The right allies for a healthy Microbiota.
Anticancer Research, 41(6), 2759–2772. https://doi.org/10.21873/
anticanres.15057
6. Shi, G., Lin, Y., Wu, Y., Zhou, J., Cao, L., Chen, J., Li, Y., Tan,
N., & Zhong, S. (2022). Bacteroides fragilis supplementation
deteriorated metabolic dysfunction, inflammation, and aorta
atherosclerosis by inducing gut Microbiota dysbiosis in animal
model. Nutrients, 14(11), 2199. https://doi.org/10.3390/nu14112199
7. Rosenfeld, D. L., Bartolotto, C., & Tomiyama, A. J. (2022).
Promoting plant-based food choices: Findings from a field
experiment with over 150,000 consumer decisions. Journal
of Environmental Psychology, 81(101825), 101825. https://doi.
org/10.1016/j.jenvp.2022.101825
8. Yang, Z., Zhu, X., Wen, A., & Qin, L. (2022). Development
of probiotics beverage using cereal enzymatic hydrolysate
fermented with Limosilactobacillus reuteri. Food Science &
Nutrition, 10(9), 3143–3153. https://doi.org/10.1002/fsn3.2913
9. Otles, S., & Nakilcioglu-Tas, E. (2022). Cereal‐based functional
foods. In: Functional Foods (pp. 55–90). Wiley. https://doi.
org/10.1002/9781119776345.ch3
10. Quispe-Herrera, R., Paredes Valverde, Y., & Roque Huamani, J.
R. (2022). Capacidad antioxidante y análisis proximal de néctar a
base de Solanum sessiliflorum y Chenopodium quinoa Willdenow.
Agronomía Mesoamericana: Órgano divulgativo del PCCMCA,
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
CONCLUSIONS
Enzymatic hydrolysis with α-amylase allowed
the hydrolysis of quinoa flour starch. Lactic acid
fermentation with 10 % inoculum and 10 hours
of fermentation was the most suitable treatment
to obtain a probiotic quinoa beverage. The
proximal analysis confirmed that the beverage has
84.6 kcal/100 g, 19.3 g carbohydrates/100 g,
0.2 g fat/100 g, and 1.4 g protein/100 g per sample.
Lactobacillus spp. is present in the beverage at
a concentration of 6.25 x 107 CFU/mL at the end
of fermentation and 3.9 x 10 6 CFU/mL two weeks
later. Therefore, the consumption of the probiotic
quinoa beverage, in the recommended doses,
would allow making the most of the nutritional
benefits of quinoa and the beneficial effects of the
probiotic microorganisms, being able to prevent the
appearance of chronic non-communicable diseases.
The benefits of consuming probiotic microorganisms
are, among many others: prevention of diarrhea,
reduction in symptoms associated with inflammatory
bowel disease, prevention of gastrointestinal
cancers, alleviation of lactose intolerance, reduction
in Helicobacter pylori infection, prevention of
intestinal inflammatory disorders and regulation
of metabolic disorders, such as serum lipids,
cholesterol levels, blood pressure and glucose
regulation (43).
It is recommended for future research to determine
the percentage of survival of probiotic cultures
over a more extended period, simulating shelf life.
Likewise, it would be convenient to evaluate the
sensory acceptance and physicochemical stability
(pH, titratable acidity) after 21 days of storage at
different temperatures, as recommended by (44).
In addition, cofactors and prebiotics that could
enhance the yield of lactic acid would be a field to
explore since, in this investigation, values of only
0.2% w/w were reached as a maximum. Furthermore,
the determination of organic acids, free amino acids,
and antioxidant activity (45), the addition or absence
of preservatives, and the behavior of the beverage
without adding additives would complement the
information on this functional quinoa beverage.
Finally, the exploration of different genera of
lactic bacteria that could improve the beverage’s
quality would be important. Weissella confusa is
recommended by Boukid set al. (46) due to its
capacity to improve viscosity and mouthfeel in
fermented products.
CONFLICT OF INTEREST
None to declare
ACKNOWLEDGEMENT
We acknowledge the Environmental Biotechnology
- Bioremediation Laboratory (Department of
Biology, Universidad Nacional Agraria La Molina),
for providing us with the facilities for the research.
AUTHORS’ CONTRIBUTIONS
CSHC wrote the manuscript and provided data for
all tables. JGJM supervised the research. All authors
reviewed the final manuscript.
REFERENCES:
1. Álvarez-Calatayud, G., Guarner, F., Requena, T., & Marcos, A. (2018).
Dieta y microbiota. Impacto en la salud. Nutrición hospitalaria:
órgano oficial de la Sociedad Española de Nutrición Parenteral y
Enteral, 35(Spec6), 11–15. https://doi.org/10.20960/nh.2280
2. Requena, T., & Velasco, M. (2021). Microbioma humano en la
salud y la enfermedad. Revista Clínica Española, 221(4), 233–240.
https://doi.org/10.1016/j.rce.2019.07.004
3. Ursa Herguedas, A. J.; Ursa Bartolomé, M. I. (2022). Manejo de
la microbiota intestinal en la prevención y tratamiento de las
enfermedades de la civilización. Medicina Naturista. 16 (1), 9-13.
4. Maza-Avila, F. J., Caneda-Bermejo, M. C., & Vivas-Castillo, A.
C. (2022). Hábitos alimenticios y sus efectos en la salud de
los estudiantes universitarios. Una revisión sistemática de la
literatura. Psicogente, 25(47), 1–31. https://doi.org/10.17081/
psico.25.47.4861
5. Divella, R., De Palma, G., Tufaro, A., Pelagio, G., Gadaleta-
Caldarola, G., Bringiotti, R., & Paradiso, A. (2021). Diet, probiotics
and physical activity: The right allies for a healthy Microbiota.
Anticancer Research, 41(6), 2759–2772. https://doi.org/10.21873/
anticanres.15057
6. Shi, G., Lin, Y., Wu, Y., Zhou, J., Cao, L., Chen, J., Li, Y., Tan,
N., & Zhong, S. (2022). Bacteroides fragilis supplementation
deteriorated metabolic dysfunction, inflammation, and aorta
atherosclerosis by inducing gut Microbiota dysbiosis in animal
model. Nutrients, 14(11), 2199. https://doi.org/10.3390/nu14112199
7. Rosenfeld, D. L., Bartolotto, C., & Tomiyama, A. J. (2022).
Promoting plant-based food choices: Findings from a field
experiment with over 150,000 consumer decisions. Journal
of Environmental Psychology, 81(101825), 101825. https://doi.
org/10.1016/j.jenvp.2022.101825
8. Yang, Z., Zhu, X., Wen, A., & Qin, L. (2022). Development
of probiotics beverage using cereal enzymatic hydrolysate
fermented with Limosilactobacillus reuteri. Food Science &
Nutrition, 10(9), 3143–3153. https://doi.org/10.1002/fsn3.2913
9. Otles, S., & Nakilcioglu-Tas, E. (2022). Cereal‐based functional
foods. In: Functional Foods (pp. 55–90). Wiley. https://doi.
org/10.1002/9781119776345.ch3
10. Quispe-Herrera, R., Paredes Valverde, Y., & Roque Huamani, J.
R. (2022). Capacidad antioxidante y análisis proximal de néctar a
base de Solanum sessiliflorum y Chenopodium quinoa Willdenow.
Agronomía Mesoamericana: Órgano divulgativo del PCCMCA,
10Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 30 | Number 01 | Article 352397Carolina S. Huapaya Castillo, Juan G. Juscamaita Morales
Programa Cooperativo Centroamericano de Mejoramiento
de Cultivos y Animales, 47706. https://doi.org/10.15517/
am.v33i2.47706
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and Lactobacillus plantarum fermentation on antioxidant activity
and metabolomic profiles of quinoa beverage. Food Research
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Ronsisvalle, S., Caggia, C., & Randazzo, C. L. (2022). Formulation
of germinated brown rice fermented products functionalized by
probiotics. Innovative Food Science & Emerging Technologies:
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of Food Science and Technology, 80(103076), 103076. https://doi.
org/10.1016/j.ifset.2022.103076
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11Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 30 | Number 01 | Article 352397
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
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https://doi.org/10.3390/foods12051005
Preparation of a probiotic quinoa beverage by enzymatic hydrolysis of its starches and subsequent lactic acid fermentation
41. Shori, A. B., & Al Zahrani, A. J. (2022). Non-dairy plant-based
milk products as alternatives to conventional dairy products for
delivering probiotics. Food Science and Technology, 42, 1-14.
https://doi.org/10.1590/fst.101321
42. Ibrahim, S. A., Ayivi, R. D., Zimmerman, T., Siddiqui, S. A.,
Altemimi, A. B., Fidan, H., Esatbeyoglu, T., & Bakhshayesh, R.
V. (2021). Lactic acid bacteria as antimicrobial agents: Food
safety and microbial food spoilage prevention. Foods (Basel,
Switzerland), 10(12), 3131. https://doi.org/10.3390/foods10123131
43. Küçükgöz, K., & Trząskowska, M. (2022). Nondairy probiotic
products: Functional foods that require more attention. Nutrients,
14(4), 753. https://doi.org/10.3390/nu14040753
44. Mousanejadi, N., Barzegar, H., Alizadeh Behbahani, B., Jooyandeh
H. (2023). Production and evaluation of a functional fruit beverage
consisting of mango juice and probiotic bacteria. Food Measure
https://doi.org/10.1007/s11694-023-01862-3
45. Lorusso A, Coda R, Montemurro M, Rizzello CG. (2018). Use of
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Novel Yogur t-Like Beverages. Foods. 7(4):51. https://doi.
org/10.3390/foods7040051
46. Boukid F, Hassoun A, Zouari A, Tülbek MÇ, Mefleh M, Aït-Kaddour
A, Castellari M. (2023). Fermentation for Designing Innovative
Plant-Based Meat and Dairy Alternatives. Foods.; 12(5):1005.
https://doi.org/10.3390/foods12051005