Food Chemistry 402 (2023) 134252

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Production of milks and kefir beverages from nuts and certain

physicochemical analysis
Emine Mine Comak Gocer *, Ebru Koptagel
Department of Nutrition and Dietetics, Faculty of Health Sciences, Akdeniz University, Antalya, Turkey

A R T I C L E I N F O A B S T R A C T

Keywords: Cashew, hazelnut, peanut, walnut, and almond were used to make nut milk and kefir in this study. Kefir starter
Nut milk culture was used to ferment cow and nut milks. Kefir samples were kept at 4 ◦ C for 30 days and examined on the
Kefir 1st, 15th, and 30th days. The highest energy contents were determined in hazelnut milk (73.71 kcal/100 g) and
Organic acids
kefir made from hazelnut milk (74.89 kcal/100 g). During storage, the total sugar, total organic acid, and
Fatty acids
Volatiles
polyunsaturated fatty acid contents of kefir samples decreased, whereas the saturated fatty acid content and pH
Physicochemical analysis value increased. The unsaturated fatty acid content of nuts milk and respective kefir was higher than cow’s milk
and kefir made from cow’s milk, while the saturated fatty acid level was lower. Volatiles, ethanol, and ethyl
acetate, which were shown to be dominating among the aroma components, were highest in kefir made from
cow’s milk.

1. Introduction
With the rise in chronic diseases such as cardiovascular, diabetes,
obesity, increased life expectancy and health-care expenses, people are
becoming more interested in functional foods, which leads to lifestyle
modifications. Newly developed food products must fulfill hunger, meet
nutritional demands, avoid chronic diseases, and leave a good impact on
both physical and mental health (Egea et al., 2020). The number of
people choosing a vegan diet has risen in recent years, owing to an in­
crease in research claiming that a vegan diet is healthier. In addition,
manufacturing of animal goods causses pollution, sensitivity is on the
rise regarding animal rights, and a dietary trend is developing in this
direction. As a result, the value of vegetable milk, which is a substitute to
animal milk and dairy products that cannot be taken as part of a vegan
diet, is growing (Erk, Seven, & Akpınar, 2019).
Nut milks are nut suspensions that look like cow’s milk when dis­
solved or broken down in water. Nut milks and fermented products
manufactured from nut milks is relatively new topic. More research into
the nutritional value of these foods is required (Mauro & Garcia, 2019).
Nuts are abundant in monounsaturated & polyunsaturated fatty acids,
vegetable proteins, dietary fiber, phytosterols, polyphenols, vitamins,
and minerals, as well as they have a great potential as a source of nut
milk. Nut milks don’t have the same nutritional value as cow’s milk;
however they have more fats. Herbal dairy products on the market
include added sugars. Consumers are demanding that these additives,
known as artificial sweeteners, be removed from products. Vegetable
milks that meet the nutritional quality and flavor of animal-derived milk
while containing no additives are expected to gain popularity (Asioli
et al., 2017). Fermented products of nut milk are preferred by consumers
for this purpose. It is well understood that the fermentation process
improves the nutritional content, aroma, taste, and texture of foods.
Fermentation produces volatile organic molecules that provide the
desired flavor and aroma (Tangyu, Muller, Bolten, & Wittmann, 2019).
Although the properties of the fermented product vary depending on the
substrate used, they generally contain sugars (sucrose, glucose, and
fructose), organic acids (lactic, acetic, citric, tartaric, butyric, malic, and
propionic acids), alcohols (ethanol, hexanol, and glycerol), and esters
(ethyl propionate, hexanoate, octanoate, and decanoate) (Fiorda et al.,
2017).
Kefir is a nutritious fermented milk beverage are probiotic. Various
reactions occur with chemicals and enzymes generated by microbes
during the multiplication of bacteria in kefir. Different metabolites such
as ethanol, CO2, and organic acids are produced as glycolytic enzymes
break down reducing carbohydrates like glucose and fructose (Destro,
Prates, Watanabe, Garcia, Biz, & Spinosa, 2019). Although cow’s milk is
the most popular source of kefir, it can also be made from the milk of

* Corresponding author at: Akdeniz University, Faculty of Health Sciences, Department of Nutrition and Dietetics, Dumlupinar Boulevard, Campus, 07058 Antalya,
Turkey.
E-mail address: minecomak@akdeniz.edu.tr (E.M. Comak Gocer).

https://doi.org/10.1016/j.foodchem.2022.134252
Received 29 April 2022; Received in revised form 30 August 2022; Accepted 11 September 2022
Available online 14 September 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
E.M. Comak Gocer and E. Koptagel
other animals such as goat, sheep, and camels, as well as from plant
sources such as soy, rice, and coconut. Individuals with cow’s milk
protein allergy and lactose intolerance sensitivity, as well as those
following a vegetarian/vegan diet, may prefer kefir made from vege­
table milk to cow’s milk kefir (Sethi, Tyagi, & Anurag, 2016).
Nuts are nutritious, it is expected that vegetable milk derived from
nuts will provide a favorable environment for fermentation and vege­
table milk derived from nuts will be suitable for kefir production.
Because the items on the market contain food additives (stabilizer,
emulsifier etc.) and sugar, they do not meet customer expectations.
There is no study that report kefir made from 100 % vegetable milk
obtained from nuts, without additives or additional sugar. Kefir was
prepared in this study by fermenting vegetable milk from almond,
peanut, hazelnut, walnut, and cashew. Cow’s milk, with a kefir starter
culture was also used. Kefir samples were kept at 4 ◦ C for 30 days. On the
1st, 15th, and 30th days of storage, the energy value, sugars, organic &
fatty acids, and volatile composition of milk and kefir samples were
determined.
2. Material and methods
2.1. Materials
Raw nuts (peanuts, hazelnuts, almonds, walnuts, and cashews) were
acquired from a local market, raw cow milk from Akdeniz University
Faculty of Agriculture’s Cattle Farm, and kefir starter culture (Kefir DC,
DuPont Danisco) from Türker Industry Teknik Makina ve Tic. Singing.
Ltd. Sti (Istanbul, Turkey). Nut milks and kefir samples were prepared in
the Department of Food Engineering of Akdeniz University, Turkey.
2.2. Milk production from nuts
Bensmira and Jiang’s method Bensmira and Jiang (2012) was
modified to make nut milk. Raw peanut (Arachis hypogaea L.), hazelnut
(Corylus avellana L.), almond (Prunus amygdalus L.), walnut (Juglans
cinerea L.), and cashew nut (Anacardium occidentale L.) were soaked in
water for 12 h and peeled. Water is added to the peeled nuts at a ratio of
1:5 (wt/v) to obtain milk with 10 g/100 g total solid content and mixed
for 10 min in a blender (Kenwood Thermoresist Glass Blender AT338).
The resulting mix was filtered by using a cloth bag (standard American
cotton cloth). The milky liquid obtained was used to produce kefir.
2.3. Kefir production from Nut’s and Cow’s milk
To make kefir, nuts & raw cow milk were separately heated at 90 ◦ C
for 5 min and then cooled to 25 ◦ C. The chilled milks were inoculated
with commercial kefir starter culture (0.015 g/L) and incubated at 25 ◦ C
until the pH reached 4.6 (Ebner et al., 2015). After incubation, kefir
samples were put into 200 mL plastic cups with lids and stored at 4 ◦ C for
30 days.
2.4. Physicochemical analysis
The fat and protein contents of milk and kefir samples were tested
using the AOAC standard method AOAC (2000). The protein content of
nut’s milks and their respective kefirs was calculated by multiplying the
total nitrogen content by 5.18. The protein content of cow milk and
respective kefir samples was calculated by multiplying the total nitrogen
content by 6.38. At 25 ◦ C, a calibrated pH meter (Orion 2 Star, Thermo
Scientific, Singapore) was used to measure the pH of milk and kefir
samples.
2.5. Determination of sugar composition
HPLC (Shimadzu LC-20AT, Tokyo, Japan) with a column (ICE-ORH-
801, 300 mm × 6.5 mm), refractive index detector (Shimadzu RID 10A,
2
Food Chemistry 402 (2023) 134252
Tokyo, Japan), and fluorescence detector (RF 20A, Tokyo, Japan) was
used to analyze lactose, fructose, glucose, and saccharose (Snyder,
Kirkland, & Glajch, 2012). The temperature in the column oven was
65 ◦ C, the injection volume was 50 μL and the flow rate was 0.6 mL/min.
5 mM H2SO4 was employed as the mobile phase in the experiment.
Analyses were carried out using the isocratic elution of 5 mM H2SO4.
The sugar profile of the samples was identified by using external stan­
dards and the total carbohydrate values of the samples were calculated
by the sum of the sugars.
2.6. Calculation of energy value
The Atwater coefficients approach [(carbohydrate × 4) + (protein ×
4) + (fat × 9)] was used to estimate the energy values of milk and kefir
samples (Maclean et al., 2003).
2.7. Determination of organic acid composition
Organic acids were quantified using UPLC-MS/MS (Dinçer, Tongur,
& Erkaymaz, 2020). 0.5 g of homogenized sample was extracted with 10
mL of water:methanol (4:1, v/v) solution containing 0.1 % (v/v) formic
acid. The mixture was vortexed for 5 min before being centrifuged for
15 min at 4 ◦ C at 1968 g. After passing the supernatant through a 0.22
µm PTFE membrane filter, 200 mL was transferred to a 2 mL vial, 800
mL of mobile phase A was added, and the sample was injected into the
UHPLC-MS/MS. The duration of the analysis was 7 min. Thermo Sci­
entific Ultimate 3000 UPLC, Thermo Scientific-TSQ Fortis (Thermo
Fisher Scientific Inc. Waltham, Massachusetts, USA) was used for the
analyses. Xcalibur software was used to perform chromatographic ana­
lyses. For chromatographic separation, a Hypersil Gold RP C18 (1.9 m),
50x2.1 mm UHPLC column (Thermo Fisher Scientific Inc. Waltham,
Massachusetts, USA) was employed. The temperature in the column
oven was 40 ◦ C, the injection volume was 10 μL, and the temperature of
MS/MS capillary was 270 ◦ C. The evaporation temperature in the
electrospray ionization module was 50 ◦ C, the auxiliary gas pressure
(Arb) was 20, the sheath gas pressure (Arb) was 50, and the spray
voltage was ± 3500 V. Mobile phase A (Water: Methanol 95:5, 0.1 %
formic acid, 4 mM ammonium formate), and mobile phase B (Methanol:
Water 95:5, 0.1 % formic acid, 4 mM ammonium formate) were used by
a gradient flow program. Elusion gradient was started with 0.25 mL/min
flow rate as 90 % mobile phase A and 10 % mobile phase B. Rate was
changed at the rate of 10 % mobile phase A and 90 % mobile phase B in
4 min, followed by 90 % mobile phase A and 10 % mobile phase B in 7
min. The system’s current mass detector is a sequential quadrupole
spectrometer, which employs three cascade quadrupoles. Soft ionization
is the ionization technique employed that is generally based on the
notion of conserving the analyte’s primary mass. It works based on
positive ionization, which involves adding a proton to the main mass (M
+ 1), or negative ionization, which involves subtracting a proton from
the main mass (M− 1) (M− 1). Quantitative results were achieved by
comparing the data collected from the device with the data produced
from analysis of the samples by examining a pure standard of organic
acids with known concentration. The limit of detection (LOD) and the
limit of quantification (LOQ) were 0.007 mg/g, and 0.02 mg/g,
respectively. For quantitative analysis, a 6-point calibration curve was
developed for each organic acid, with concentration values of 0.02,
0.10, 0.50, 2.0, and 5.0 mg/g.
2.8. Determination of fatty acids composition
The composition of fatty acids was determined using GC–MS. By
reacting sodium hydroxide and methanol with 0.1 g sample according to
AOCS Official Method Ce 1 h-05 (AOCS, 2009), fatty acids in kefir
samples are transformed into fatty acids methyl esters (FAME). The
sample was mixed with 10 mL n-Hexane and vortexed for 5 min. The
supernatant was injected into GC–MS at 1/50 dilution with acetone into
E.M. Comak Gocer and E. Koptagel
a 2 mL vial after centrifugation of the entire mixture. The duration of the
analysis was 63 min.
Thermo Scientific Trace 1300 GC and Thermo Scientific ISQ7000
single Quadrupole Mass Spectrometer (Thermo Fisher Scientific Inc.
Waltham, Massachusetts, USA) system were used for the analyses.
Xcalibur software was used to perform chromatographic analyses. For
chromatographic separation, a TraceGOLD TG-624SilMS GC UHPLC
column (Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA)
was employed. Based on Saturn spectra published in the Willey 1n.l and
NIST 0.5 (National Institute of Standards and Technology) databases,
compounds were identified by comparison. For each identified chemi­
cal, major, molecular, and qualifying ions were chosen. The inlet tem­
perature was 250 ◦ C, and injection volume was 2 μL with 1/50 split
ratio. The carrier gas was helium, and the flow rate was 1.5 mL/min. The
oven temperature was increased to 300 ◦ C a rate of 5 ◦ C/min after 3 min
of holding at 50 ◦ C and was held at 300 ◦ C for 10 min. The temperature
of the detector was 280 ◦ C. Quantitative results were achieved by
comparing the data obtained from the device with the data obtained
from the examination of the samples by analyzing a pure standard of
fatty acids methyl esters (FAME, Supelco, Bellefonte, PA, USA) with
known concentration. The LOD was 20 mg/100 g, while the LOQ was 50
mg/100 g.
2.9. Determination of volatile composition
GC–MS was used to do quantitative analysis of volatile components.
0.5 g homogenized sample was extracted with 10 mL acetone solvent,
according to the Kıvrak, & Kıvrak (2018). After homogenizing the
mixture with ultra-turrax for 15 min, it was centrifuged for 15 min at
4000 rpm. Supernatant was filtered through a 0.22 m PTFE membrane
Fig. 1. Physicochemical properties and nutrients of milk and kefir samples. a–f: letters for each property and factor shows significant different by Duncan’s
multiple range test (p < 0.05).
3
Food Chemistry 402 (2023) 134252
filter and fed into the GC–MS at 1/5 dilution with acetone into a 2 mL
vial. Total analysis time was 28 min. Thermo Scientific Trace 1300 GC
and Thermo Scientific ISQ7000 single Quadrupole Mass Spectrometer
(Thermo Fisher Scientific Inc. Waltham, Massachusetts, USA) system
were used for the analyses. Xcalibur software was used to perform
chromatographic analyses. For chromatographic separation, a Trace­
GOLD TG-624SilMS GC UHPLC column (Thermo Fisher Scientific Inc.
Waltham, Massachusetts, USA) was employed. Based on Saturn spectra
published in the Willey 1n.l and NIST 0.5 (National Institute of Stan­
dards and Technology) databases, compounds were identified by com­
parison. For each identified chemical, major, molecular, and qualifying
ions were chosen. The device’s inlet temperature was 250 ◦ C and the
injection volume was 10 μL with a 1/5 split ratio. The carrier gas was
helium, and the flow rate was 1.5 mL/min. Oven temperature program:
Hold 3 min at 30 ◦ C, increased to 230 ◦ C at a rate of 10 ◦ C/min and
maintained for 5 min. The temperature of the detector was 280 ◦ C. The
volatiles were quantified using an ethanol calibration curve as a typical
volatile component. A 6-point calibration curve for ethanol was
employed for quantitative analysis, with concentration values of 1, 5,
10, 50, 100, and 500 mg/L.
2.10. Statistical analyses
The experiment was carried out in two replications, with the analyses
running concurrently. The averages of analyses were subjected to
analysis of variance using the SAS statistical program (SAS Institute Inc.
SAS System 9.0). Duncan Multiple Comparison Test was used to evaluate
the effect levels of the sources of variation that were determined to be
significant.
E.M. Comak Gocer and E. Koptagel Food Chemistry 402 (2023) 134252

3. Results and discussion 3.2. Sugar profile

3.1. Physicochemical properties
Fig. 1a and c show the carbohydrate, protein, fat, and energy con­
tents of milk samples. Cow’s milk has the highest carbohydrate and
protein content, while walnut milk has the highest fat content (p < 0.05)
(Table S1). All nut milks have been found to have higher energy than
cow’s milk. This argument is supported by the fact that nut milks had
higher fat contents than cow’s milk. Indeed, fat stores more energy than
carbohydrates and proteins (Isanga & Zhang, 2009). Cow milk had the
lowest energy value (p < 0.05) while hazelnut milk had the highest
energy value (p < 0.05). The highest carbohydrate and protein values
were determined in kefir produced from cow’s milk while the highest fat
value was determined in kefir produced from walnut milk (p < 0.05)
(Fig. 1b). When the energy values of kefir samples were compared, the
kefir made from hazelnut milk had the highest energy value, whereas
kefir made from cow’s milk had the least (p < 0.05) (Fig. 1c). The
highest pH value was found in hazelnut milk, while the lowest pH value
was found in cashew milk (p < 0.05) among the nut milk samples
(Fig. 1a, Table S2).
As shown in Table 1, kefir sample made from walnut milk had the
highest pH value, while the kefir made from cashew milk had the lowest
pH. The pH of fermented items is said to be generally retained or
reduced during storage. According to a report, lactose continues to be
broken down by bacteria during storage into lactic acid, which is the
cause of this pH drop (Guzel-Seydim et al., 2005). On the contrary, in
our study the pH values of the samples increased (p < 0.05) during
storage. This scenario is thought to have emerged because of the usage of
organic acids such as lactic, malic, oxalic, and citric acids as an energy
source and a defensive mechanism for the yeast and bacteria in the kefir
starting culture to survive in acidic environments (Cirlini, Ricci, Gal­
averna, & Lazzi, 2020).
Sugar profile determination during fermentation and storage in fer­
mented plant products is significant because it offers information on
microbial metabolic activities. Fig. 2a shows the sugar profile of nut milk
samples. Almond milk had the highest glucose level, while cashew milk
had the highest fructose and sucrose contents (p < 0.05) (Table S2).
While the lactose value of cow’s milk was 3.90 ± 0.06 g/100 g, lactose
was 2.28 ± 0.16 and galactose was 0.60 ± 0.34 in kefir made from cow’s
milk on the first day of storage while on the 30th day of storage, the
lactose and galactose values were 1.56 ± 0.14 and 0.18 ± 0.27 (p <
0.05), respectively. Kefir made from nut milk was lactose-free, making it
suitable for lactose-intolerant individuals. In our study, cashew kefir had
the greatest glucose and fructose levels, while kefir made from almond
milk had the highest sucrose level (p < 0.05). The glucose, fructose, and
sucrose concentrations in the samples reduced (p < 0.05) after storage
(Table 1). During fermentation, microorganisms consume carbohydrates
as an energy source. Both fermentation and storage result in a decrease
in total sugar content. This is due to microbes’ usage of sugar to maintain
the required metabolic activity (Łopusiewicz et al., 2019). While bac­
teria in cow’s milk use lactose as their only source of carbon, bacteria in
nut milks use glucose or sucrose as an energy source and fructose as an
electron acceptor (Ortiz, Fornaguera, Raya, & Mozzi, 2012). Yeasts
make glucose and fructose by hydrolyzing sucrose with the enzyme
invertase (β-D-fruktofuranosidaz), which they release. While glucose and
fructose become available to bacteria, they are absorbed into the cell by
facilitated diffusion through yeast cells and used in metabolic activities
(Lynch, Wilkinson, Daenen, & Arendt, 2021). Yeasts can use a variety of
dietary components as carbon and energy sources, but they prefer
glucose and fructose (Casal, Paiva, Queirós, & Soares-Silva, 2008). Ac­
cording to our findings, the proportion of glucose and fructose measured
in the composition of nut milks reduced more than sucrose because of
fermentation due to yeast activity, as expected.

Table 1
Effect of the sample type and storage time on physicochemical properties of kefir samples.
Factor pH Glucose (g/ Fructose (g/ Saccharose (g/ Total carbohydrate Total organic SFA (mg/ 100 MUFA (mg/ PUFA (mg/
100 mL) 100 mL) 100 mL) (g/100 mL) acid (mg/g) g) 100 g) 100 g)

Type of sample
Cow 4.82 ± 0.00 ± 0.00f 0.00 ± 0.00f 0.00 ± 0.00f 2.17 ± 0.40a 21.90 ± 1.89b 23224.33 ± 10007.63 ± 1609.07 ±
0.11d 1254.35a 1415.32f 141.01f
Cashew 4.76 ± 2.42 ± 0.51a 5.33 ± 0.61a 3.04 ± 0.87b 1.07 ± 0.19b 31.25 ± 3.46a 11209.03 ± 31291.87 ± 9492.63 ±
0.06e 135.44b 2283.01d 108.88c
Hazelnut 4.89 ± 1.63 ± 0.34c 2.31 ± 0.47c 1.26 ± 0.28c 0.51 ± 0.10d 12.49 ± 1.85d 7757.38 ± 61627.74 ± 6510.39 ±
0.16c 602.63d 3335.42a 518.03d
Peanut 4.97 ± 1.16 ± 0.58d 0.76 ± 0.92e 0.99 ± 0.18d 0.28 ± 0.16e 18.44 ± 1.87c 9519.47 ± 48753.97 ± 3254.37 ±
0.16b 422.65c 2295.83b 118.00e
Walnut 5.32 ± 0.95 ± 0.20e 0.93 ± 0.46d 0.43 ± 0.37e 0.23 ± 0.09f 6.07 ± 1.36e 11207.96 ± 12740.60 ± 58237.70 ±
0.17a 902.42b 1558.70e 408.41a
Almond 4.84 ± 2.11 ± 1.20b 3.08 ± 2.04b 4.10 ± 0.79a 0.93 ± 0.40c 21.09 ± 3.33b 6715.87 ± 36582.50 ± 18141.01 ±
0.03dc 372.76e 1242.35c 363.05b

Storage time (days)

1 4.78 ± 2.02 ± 1.24a 3.04 ± 2.15a 2.17 ± 1.83a 1.19 ± 0.84a 21.71 ± 8.20a 11533.21 ± 33466.37 ± 16279.70 ±
0.14c 5609.28a 18396.14b 9684.67a
15 4.98 ± 1.16 ± 0.69b 1.83 ± 1.96b 1.58 ± 1.41b 0.77 ± 0.60b 18.21 ± 7.02b 11541.07 ± 33312.48 ± 16228.22 ±
0.18b 5449.80b 18378.96c 9590.16b
30 5.05 ± 0.95 ± 0.58c 1.33 ± 1.52c 1.15 ± 1.13c 0.63 ± 0.54c 15.71 ± 8.79c 11742.73 ± 33723.30 ± 16114.66 ±
0.23a 5309.02c 18289.45a 4937.85c

ANOVA
TP *** *** *** *** *** *** *** *** ***
ST *** *** *** *** *** *** *** *** **
TP × ST ** *** *** *** *** ** *** *** ***

a-f letters for each column and factor are significantly different by Duncan’s multiple range test (p < 0.05); for ANOVA,** and *** denotes significant difference of P <
0.01 and P < 0.001, respectively. SFA: Saturated fatty acid, MUFA: Monounsaturated fatty acid, PUFA: Polyunsaturated fatty acid.

4
E.M. Comak Gocer and E. Koptagel Food Chemistry 402 (2023) 134252

Fig. 2. Sugar profile and fatty acid profile of milk samples. a–f: letters for each property and factor shows significant different by Duncan’s multiple range test (p
< 0.05). SFA: Saturated fatty acid, MUFA: Monounsaturated fatty acid, PUFA: Polyunsaturated fatty acid.

3.3. Organic acid profile highest levels of malic acid.

Depending on the type and amount of organic acids, chemical
qualities such as pH, total acidity, and volatile organic compounds can
alter the sensory properties of foods (Liszkowska & Berlowska, 2021). A
group of microorganisms in kefir may emit an organic acid that another
group of bacteria in kefir may use as a substrate since kefir has a complex
flora. This could affect the production of other metabolites, which in
turn could affect the sensory properties of the finished product (Bulat &
Topcu, 2021). As a result, its examination is critical. The organic acid
profile of milk samples is shown in Table S3. Cashew milk has the
highest total organic acid content, whereas cow’s milk has the lowest
total organic acid content (Fig. 1a). In cow’s milk, only lactic and citric
acids were found. Cashew and almond milks had the highest levels of
oxalic acid, whereas hazelnut, peanut, and walnut milks contained the
Organic acids are generated during carbohydrate fermentation, lipid
hydrolysis, and microbial activity during food production and storage.
Once synthesized, acids can be used to form other aromatic compounds
such as ketones, alcohols, aldehydes, and esters (Batista et al., 2017). A
total of 11 organic acids were found in kefir samples, but acetic acid and
propionic acid were not detected in nuts milk kefir samples (Table 2). In
the production of kefir from cow’s milk, lactic acid, ethanol, and acetic
acid fermentation occurs during fermentation. After lactose is broken
down into glucose and galactose, lactic acid bacteria metabolize glucose
homofermentatively (Hikmetoglu, Sogut, Sogut, Gokirmaklı, & Guzel-
Seydim, 2020). Lactic acid is one of the most important end products
of fermentation in kefir made from cow’s milk. Lactic acid causes an
acidic taste in the product and, together with volatile organic com­
pounds, gives the aroma of kefir (Atalar, 2019). In our study, lactic acid

Table 2
Organic acid profile of kefir samples (mg/g organic acid by product).
Organic acids Storage time (days) Cow Cashew Hazelnut Peanut Walnut Almond

Formic acid 1 2.21 ± 0.23 0.09 ± 0.03 0.02 ± 0.01 0.04 ± 0.01 n.d 0.09 ± 0.03
15 1.84 ± 0.01 0.05 ± 0.01 n.d 0.02 ± 0.01 n.d 0.08 ± 0.02
30 1.68 ± 0.33 0.04 ± 0.02 n.d n.d n.d 0.05 ± 0.01
Acetic acid 1 0.08 ± 0.01 0.03 ± 0.01 n.d n.d n.d 0.02 ± 0.01
15 0.97 ± 0.12 n.d n.d n.d n.d n.d
30 1.21 ± 0.15 n.d n.d n.d n.d n.d
Propionic acid 1 0.08 ± 0.01 n.d n.d n.d n.d n.d
15 0.07 ± 0.01 n.d n.d n.d n.d n.d
30 0.07 ± 0.02 n.d n.d n.d n.d n.d
Succinic acid 1 0.78 ± 0.09 1.00 ± 0.14 0.76 ± 0.13 1.17 ± 0.14 0.11 ± 0.04 0.92 ± 0.11
15 0.63 ± 0.11 0.95 ± 0.03 0.66 ± 0.09 0.84 ± 0.13 n.d 0.96 ± 0.06
30 0.47 ± 0.06 0.89 ± 0.09 0.34 ± 0.04 0.84 ± 0.02 n.d 0.84 ± 0.04
Lactic acid 1 18.89 ± 2.64 0.29 ± 0.02 0.07 ± 0.01 0.13 ± 0.01 n.d 0.06 ± 0.01
15 15.17 ± 1.05 0.25 ± 0.01 0.08 ± 0.02 0.07 ± 0.01 n.d 0.04 ± 0.01
30 16.31 ± 0.92 0.22 ± 0.01 0.05 ± 0.01 0.07 ± 0.02 n.d 0.02 ± 0.01
Oxalic acid 1 0.04 ± 0.01 12.12 ± 1.08 5.10 ± 0.63 4.15 ± 0.44 2.10 ± 0.43 10.85 ± 1.09
15 n.d 10.20 ± 1.13 4.42 ± 0.17 4.06 ± 0.23 1.94 ± 0.22 9.93 ± 0.83
30 n.d 9.67 ± 0.54 4.53 ± 0.89 4.06 ± 0.57 n.d 6.55 ± 0.71
Citric acid 1 1.90 ± 0.12 4.72 ± 0.36 1.32 ± 0.25 1.98 ± 0.06 1.76 ± 0.24 1.06 ± 0.05
15 1.68 ± 0.27 4.20 ± 0.23 1.38 ± 0.11 1.89 ± 0.03 1.68 ± 0.16 0.82 ± 0.07
30 1.49 ± 0.23 3.95 ± 0.18 1.32 ± 0.06 1.89 ± 0.05 n.d 0.82 ± 0.12
Fumaric acid 1 0.04 ± 0.01 0.18 ± 0.01 0.09 ± 0.02 0.12 ± 0.01 n.d 0.19 ± 0.03
15 n.d 0.23 ± 0.04 0.06 ± 0.01 0.09 ± 0.01 n.d 0.23 ± 0.01
30 n.d 0.18 ± 0.02 0.04 ± 0.01 0.09 ± 0.02 n.d 0.23 ± 0.02
Malic acid 1 n.d 14.06 ± 3.35 5.49 ± 0.28 11.75 ± 1.05 5.89 ± 1.05 10.02 ± 1.09
15 n.d 11.62 ± 2.16 4.01 ± 0.17 9.11 ± 0.93 4.46 ± 0.82 7.03 ± 1.07
30 n.d 10.87 ± 2.48 2.72 ± 0.06 9.11 ± 1.08 n.d 7.03 ± 0.89
Tartaric acid 1 n.d 0.12 ± 0.01 0.08 ± 0.03 0.09 ± 0.02 n.d 0.05 ± 0.04
15 n.d 0.11 ± 0.03 0.08 ± 0.01 0.11 ± 0.01 n.d 0.03 ± 0.01
30 n.d 0.07 ± 0.01 0.09 ± 0.02 0.11 ± 0.03 n.d 0.03 ± 0.01
Benzoic acid 1 0.09 ± 0.02 2.93 ± 0.12 1.85 ± 0.14 1.39 ± 0.25 0.18 ± 0.01 1.92 ± 0.35
15 n.d 2.60 ± 0.54 1.49 ± 0.10 1.06 ± 0.14 0.09 ± 0.01 1.71 ± 0.20
30 n.d 2.13 ± 0.31 1.39 ± 0.67 1.06 ± 0.20 n.d 1.71 ± 0.14

5
E.M. Comak Gocer and E. Koptagel
was the major acid in kefir samples made from cow’s milk, while malic
acid was dominant in kefir samples made from nut milks. While malic
acid has the most impact on the flavor of nut kefir, lactic acid has the
greatest impact on the flavor of cow kefir. On the first day of storage, the
highest malic acid value (14.06 mg/g) was found in kefir made from
cashew milk (Table 2). As a result of fermentation, the amount of oxalic
acid and malic acid identified in nut milks rose in their respective kefir
samples. Propionic acid, which has an unpleasant taste, was not found in
milk samples, and only minimal levels were found in kefir made from
cow’s milk after fermentation. Only kefir samples made from cow’s milk
included acetic acid, which was not found in milk samples or kefir
samples made from nuts milk, in general. Although acetic acid is a
byproduct of citrate metabolism, it is also produced in hetero­
fermentative lactose metabolism by the oxidation of lactic acid. The
oxidation of ethanol by acetic acid bacteria can also create it. Because
acetic acid gives items a vinegar flavor, it’s best to keep it to a minimum
in fermented foods (Chaiyasut, Makhamrueang, Peerajan, & Sivamar­
uthi, 2017. The absence of acetic acid in the samples produced from nut
milks during storage in our investigation implies that acetic acid was not
produced as a result of the activity of neither yeasts nor acetic acid
bacteria. Fermentation raised the levels of citric acid, benzoic acid, and
succinic acid in all kefir samples. The principal organic acid produced by
yeasts is succinic acid. It is produced when isocitrate is oxidized in the
glyoxylate cycle, but it can also be found in the citric acid cycle. Succinic
acid is an organic acid with a salty and bitter taste, and whose presence
in high levels may adversely affect the taste of the product (Martinez,
Bressani, Dias, Simão, & Schwan, 2019). Tartaric acid, on the other
hand, increased during fermentation in all other kefir samples except
those made from cow and walnut milks (Table 2). The metabolism of
tartaric acid in bacteria has not been thoroughly studied (Cirlini, Ricci,
Galaverna, & Lazzi, 2020). Lactic acid levels in kefir made from cow’s
milk dropped during storage in our study. During storage, lactic acid was
not determined in walnut milk kefir samples, but it was detected in
minimal amounts in all other kefir samples (Table 2). The amount of
lactic acid produced is determined by the type of lactic acid bacteria
used and the content of the fermentation medium. Lactic acid bacteria
can make it from sugar metabolism as well as from metabolizing poly­
alcohols like glycerol or acids like malic acid (Cirlini, Ricci, Galaverna,
& Lazzi, 2020).
When the total organic acid values of the kefir samples were
analyzed, the kefir made from cashew milk had the highest total organic
acid content, whereas kefir made from walnut milk had the lowest (p <
0.05). The total organic acid levels of the samples dropped (p < 0.05)
during storage (Table 1). Lactic acid bacteria’s major function in the
fermentation process is to make organic acids from sugars in the envi­
ronment, either homofermentatively or heterofermentatively. García-
Quintáns et al. (2008) found that these microbes can also metabolize
organic acids. Volatile organic compounds are formed because of the
usage of organic acids by lactic acid bacteria, which have a favorable
effect on the quality of the final goods (Li et al., 2021). Malic and citric
acids are known to be easily digested anaerobically by lactic acid bac­
teria (Mendes Ferreira & Mendes-Faia, 2020). Organic volatiles such as
diacetyl, acetoin, butanediol, acetate, and acetaldehyde are generated as
a result of microbial breakdown of citric acid and can have considerable
effects on the odors of fermented food products (Smid & Kleerebezem
2014). The metabolic processes by which yeasts and lactic acid bacteria
metabolize organic acids are well understood. According to Suárez-Lepe
and Morata (2012), Saccharomyces cerevisiae can both generate and
metabolize malic acid. Furthermore, organic acids produced by micro­
bial activity can be used as substrates in the biosynthesis of esters
(Mendes Ferreira & Mendes-Faia, 2020). The metabolic activities of
microorganisms were found to be the cause of the decrease in total
amount of organic acid during storage in our study.
None of the analyzed organic acids could be detected in the kefir
sample produced from walnut milk on the 30th day of storage (Table 2).
Carbonic acid was suspected as the reason of the pH value found in a
6
Food Chemistry 402 (2023) 134252
kefir sample made from walnut milk on the 30th day of storage.
Hydrogen ions are separated, and carbonic acid is formed as a result of
the reaction of carbon dioxide produced by yeasts due to the breakdown
of carbohydrates with water, increasing the acidity of the environment
(Mitchell, Jensen, Cliffe, & Maroto-Valer, 2010; Ge et al., 2020).
3.4. Fatty acid profile
Table S4 shows the values of saturated, monounsaturated, and
polyunsaturated fatty acids in milk samples. Saturated fatty acids such
as palmitic, heptadecanoic, and stearic acids were found in all milk
samples. Only cow’s milk included butyric, caproic, caprylic, lauric, and
pentadecanoic acids, although arachidic acid was found in all milk
samples except cow’s milk. Only cashew and peanut milk included
behenic and lignoceric acids. Undecanoic acid was found only in cashew
and walnut milk, while capric acid was found only in cow’s and walnut
milk. Except for peanut milk, myristic acid was found in all milk sam­
ples. Monounsaturated fatty acids, myristoleic and cis-10-heptadecenoic
acids, were found solely in cow’s milk, while erucic acid was found only
in peanut milk. All the milk samples contained palmitoleic and oleic
acids. Almond milk had the highest level of palmitoleic acid, while
hazelnut milk had the highest level of oleic acid. All milk samples
included linoleic acid, a polyunsaturated fatty acid, while -linolenic acid
was found in all samples except cow’s milk. As shown in Fig. 2b, cow’s
milk had the maximum saturated fatty acid value, hazelnut milk had the
highest monounsaturated fatty acid value, and walnut milk had the
highest polyunsaturated fatty acid value (p < 0.05) (Table S2).
The saturated fatty acids, palmitic, heptadecanoic, and stearic acids,
were found in all kefir samples (Table 3), whereas arachidic acid was not
detected in cow’s milk kefir. Butyric, caproic, caprylic, lauric, and
pentadecanoic acids were only found in cow’s milk kefir, while behenic
and lignoceric acids were only found in kefir made from cashew and
peanut milks. Myristic acid was detected in all samples except kefir
produced from peanut milk. Only samples made from cow’s milk and
walnut milk included capric acid, while samples made from cashew milk
and walnut milk had undecanoic acid. None of the kefir samples con­
tained tridecanoic, henicosanoic, or trichosanoic acids. Mono­
unsaturated fatty acids, myristoleic and cis-10-heptedecenoic acids,
were found only in cow’s milk kefir, but palmitoleic and oleic acids from
omega-9 fatty acids were found in all kefir samples. Only kefir made
from peanut milk contained erucic acid, an omega-9 fatty acid. None of
the kefir samples included elaidic, cis-10-pentadecenoic, cis-11-eicose­
noic, or nervonic acids. On the 30th day of storage, the maximum oleic
acid was found in kefir made from hazelnut milk, while the lowest
amount was in kefir made from cow’s milk on the 15th day of storage
(Table 3). When the polyunsaturated fatty acids of kefir samples were
examined, linoleic acid, one of the omega-6 fatty acids, was detected in
all kefir samples, while α-linolenic acid, one of the omega-3 fatty acids,
was detected in kefir produced from all nut milks, except kefir produced
from cow’s milk. Walnut kefir on the 15th day of storage had the largest
quantity of linoleic acid, while on the 1st day of storage, it had the
highest amount of α-linolenic acid (Table 3).
As shown in Table 1, kefir made from cow’s milk had the highest
total saturated fatty acids, kefir made from hazelnut milk had the
highest total monounsaturated fatty acids, and kefir made from walnut
milk had the highest total polyunsaturated fatty acids (p < 0.05). Kefir
made from almond milk had the lowest total saturated fatty acid, while
kefir made from cow milk had the lowest total mono- and poly­
unsaturated fatty acids (p < 0.05). The total saturated fatty acids values
of the samples during storage did not change significantly (p > 0.05).
The total monounsaturated fatty acids values, on the other hand,
dropped between the 1st and 15th days while increased between the
15th and 30th days (p < 0.05). The decrease in the total poly­
unsaturated fatty acids in kefir samples was found to be significant (p <
0.05) between the 15th and 30th days of storage.
The principal saturated fatty acids found in plant-based milks are
E.M. Comak Gocer and E. Koptagel Food Chemistry 402 (2023) 134252

Table 3
Fatty acid profile of kefir samples during various storage times (mg/100 g fatty acid by product).
Fatty acids Storage time (days) Cow Cashew Hazelnut Peanut Walnut Almond

C 4:0 1 831.45 ± 33.01 n.d n.d n.d n.d n.d

15 842.17 ± 45.08 n.d n.d n.d n.d n.d
30 885.64 ± 57.23 n.d n.d n.d n.d n.d
C 6:0 1 589.90 ± 29.72 n.d n.d n.d n.d n.d
15 573.62 ± 20.08 n.d n.d n.d n.d n.d
30 559.20 ± 43.95 n.d n.d n.d n.d n.d
C 8:0 1 455.83 ± 27.30 n.d n.d n.d n.d n.d
15 460.98 ± 59.02 n.d n.d n.d n.d n.d
30 437.56 ± 42.88 n.d n.d n.d n.d n.d
C 10:0 1 831.23 ± 65.55 n.d n.d n.d 148.81 ± 9.87 n.d
15 822.38 ± 61.30 n.d n.d n.d 187.87 ± 12.36 n.d
30 916.15 ± 62.97 n.d n.d n.d 209.38 ± 15.43 n.d
C 11:0 1 n.d 151.85 ± 11.20 n.d n.d 355.94 ± 21.47 n.d
15 n.d 140.35 ± 13.19 n.d n.d 373.55 ± 21.70 n.d
30 n.d 132.93 ± 9.25 n.d n.d 357.39 ± 26.84 n.d
C 12:0 1 872.11 ± 15.29 n.d n.d n.d n.d n.d
15 981.55 ± 63.21 n.d n.d n.d n.d n.d
30 944.11 ± 67.90 n.d n.d n.d n.d n.d
C 13:0 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 14:0 1 4477.90 ± 125.36 81.14 ± 8.23 22.27 ± 0.96 n.d 109.65 ± 8.71 36.79 ± 1.90
15 4530.22 ± 409.78 80.21 ± 5.17 22.09 ± 3.87 n.d 108.95 ± 10.06 37.03 ± 2.23
30 4796.66 ± 207.69 81.80 ± 10.69 22.36 ± 1.15 n.d 110.07 ± 10.99 36.68 ± 5.67
C 14:1 1 563.09 ± 234.18 n.d n.d n.d n.d n.d
15 587.09 ± 96.34 n.d n.d n.d n.d n.d
30 572.87 ± 46.90 n.d n.d n.d n.d n.d
C 15:0 1 528.23 ± 30.07 n.d n.d n.d n.d n.d
15 572.61 ± 33.21 n.d n.d n.d n.d n.d
30 518.65 ± 62.85 n.d n.d n.d n.d n.d
C 15:1 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 16:0 1 9438.44 ± 250.87 5633.45 ± 250.12 4728.60 ± 264.31 4939.96 ± 185.55 7099.55 ± 603.10 5326.20 ± 321.08
15 9337.95 ± 369.08 5865.59 ± 107.39 4689.55 ± 147.30 4847.50 ± 536.24 7553.18 ± 554.87 5160.10 ± 195.55
30 9267.22 ± 125.40 5981.89 ± 690.10 4748.02 ± 563.98 4896.87 ± 451.20 8027.71 ± 140.83 5410.67 ± 473.91
C 16:1 1 300.31 ± 20.89 273.85 ± 46.60 151.35 ± 21.93 41.46 ± 3.87 60.49 ± 2.25 474.70 ± 23.21
15 237.12 ± 14.57 290.72 ± 18.79 110.43 ± 8.74 74.69 ± 3.49 57.04 ± 1.97 447.38 ± 36.09
30 264.87 ± 12.46 376.09 ± 26.28 151.81 ± 8.50 53.10 ± 2.17 70.76 ± 6.80 403.47 ± 51.24
C 17:0 1 262.77 ± 27.35 60.86 ± 5.70 37.12 ± 2.58 41.46 ± 3.40 39.16 ± 2.23 42.92 ± 15.87
15 259.98 ± 18.66 47.16 ± 3.02 61.81 ± 1.33 63.69 ± 5.66 38.91 ± 1.07 83.21 ± 26.17
30 258.01 ± 31.63 91.35 ± 7.34 37.27 ± 2.95 41.10 ± 2.21 39.31 ± 4.36 62.79 ± 3.45
C 17:1 1 187.15 ± 10.09 n.d n.d n.d n.d n.d
15 236.92 ± 14.25 n.d n.d n.d n.d n.d
30 205.35 ± 1.98 n.d n.d n.d n.d n.d
C 18:0 1 5286.25 ± 63.45 4731.55 ± 201.98 2980.22 ± 136.90 2003.44 ± 98.26 2878.59 ± 145.20 1207.97 ± 45.74
15 4735.30 ± 121.08 4677.43 ± 539.80 2856.43 ± 195.22 2162.19 ± 144.20 2661.57 ± 203.30 1215.96 ± 69.38
30 4399.43 ± 362.48 4370.17 ± 150.10 2692.05 ± 233.08 2484.21 ± 88.16 2588.93 ± 96.61 1404.31 ± 100.44
C 18:1n9c 1 8706.42 ± 135.94 31011.16 ± 1258.74 61575.74 ± 3009.58 48320.85 ± 1005.89 12688.06 ± 1438.21 36361.20 ± 1354.20
15 8613.73 ± 407.20 30656.50 ± 965.30 61067.23 ± 1005.69 48986.45 ± 3697.14 12607.45 ± 996.25 35808.26 ± 2710.03
30 9548.48 ± 550.67 31264.30 ± 573.88 61828.66 ± 6022.96 48485.36 ± 2201.39 12737.01 ± 1130.77 36248.00 ± 2894.64
C 18:1n9t 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 18:2n6c 1 1523.02 ± 104.05 9290.67 ± 543.80 6213.25 ± 350.41 2292.28 ± 197.25 46932.68 ± 2514.22 17911.06 ± 905.64
15 1806.81 ± 96.30 9184.42 ± 706.63 6161.94 ± 108.90 2249.38 ± 86.34 47028.16 ± 3741.08 18029.50 ± 1084.26
30 1495.39 ± 86.74 9366.51 ± 292.44 6238.77 ± 763.72 2272.29 ± 120.20 46117.61 ± 1698.36 17856.79 ± 1217.16
C 18:2n6t 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 18:3n6 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 18:3n3 1 n.d 192.28 ± 10.60 318.89 ± 40.91 1060.26 ± 50.01 11721.64 ± 800.09 221.69 ± 30.06
15 n.d 180.43 ± 7.01 346.01 ± 12.04 840.41 ± 25.66 11350.98 ± 658.56 192.83 ± 18.14
30 n.d 263.61 ± 15.48 250.33 ± 19.74 1051.01 ± 79.37 11564.54 ± 521.70 211.17 ± 25.10
C 20:0 1 n.d 379.49 ± 26.89 123.93 ± 10.86 510.09 ± 33.30 211.47 ± 19.04 36.79 ± 6.02
15 n.d 315.84 ± 27.06 103.07 ± 9.03 498.67 ± 60.83 270.12 ± 21.38 47.03 ± 3.60
30 n.d 422.10 ± 50.01 144.35 ± 7.22 604.77 ± 20.06 252.28 ± 20.05 36.68 ± 1.09
C 20:1n9 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 20:2 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
(continued on next page)

7
E.M. Comak Gocer and E. Koptagel Food Chemistry 402 (2023) 134252

Table 3 (continued )
Fatty acids Storage time (days) Cow Cashew Hazelnut Peanut Walnut Almond

30 n.d n.d n.d n.d n.d n.d

C 20:3n6 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 20:3n3 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 20:4n6 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 20:5n3 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 21:0 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 22:0 1 n.d 60.86 ± 5.59 n.d 914.95 ± 60.20 n.d n.d
15 n.d 60.16 ± 4.05 n.d 1090.34 ± 103.77 n.d n.d
30 n.d 61.35 ± 4.27 n.d 1303.48 ± 100.04 n.d n.d
C 22:1n9 1 n.d n.d n.d 82.93 ± 5.80 n.d n.d
15 n.d n.d n.d 91.37 ± 9.79 n.d n.d
30 n.d n.d n.d 122.20 ± 10.35 n.d n.d
C 22:2 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 22:6n3 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 23:0 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
C 24:0 1 n.d 65.93 ± 3.08 n.d 658.17 ± 25.02 n.d n.d
15 n.d 65.17 ± 6.10 n.d 743.98 ± 38.65 n.d n.d
30 n.d 66.47 ± 9.93 n.d 751.56 ± 54.81 n.d n.d
C 24:1n9 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d

n.d. not detected.

palmitic and stearic acids; unsaturated fatty acids include oleic, linoleic,
and -linolenic acids. Individuals who consume unsaturated fatty acids
such as oleic acid instead of saturated fatty acids in their diet have
higher HDL (high-density lipoprotein) cholesterol, lower LDL (low-
density lipoprotein) cholesterol, lower triacylglycerol, and lower lipid
oxidation. Unsaturated fatty acids have beneficial effects on Alzheimer’s
disease as well as they lower blood lipid levels. Monounsaturated fatty
acids have been shown to have anti-carcinogenic benefits based on data
gained in experimental animals and human cell culture investigations
(Isanga & Zhang, 2009). In this regard, it is expected that our nut milks
and their respective kefirs would have a beneficial effect on health when
consumed.
3.5. Organic volatile profile
The aroma of nut milk is affected by both the type of seeds/nuts and
the production process applied. Aroma is a mix of taste and smell that
plays an essential part in a product’s consumer acceptance. Food flavor
is linked to the profile of volatile chemicals. The most prevalent volatile
chemical compounds discovered in nuts include aldehydes, alcohols,
ketones, alkanes, and terpenes (Manousi & Zachariadis, 2019). Table S5
shows the volatile organic compounds determined in milk samples. It
can be noted that all milk samples included 2 methyl butanal, 3 methyl
butanal, and hexanal. Only cow’s milk contained acetaldehyde and
diacetyl, while all other milk included octenal, nonanal, and benzalde­
hyde. The chemicals, 1-hexanol, heptanal, octanal, and a-pinene were
exclusively detected in walnut milk, while heptanal, octanal, and
α-pinene were only found in peanut milk. Except for peanut milk, all
samples included heptanal and pentanal.
Organic acids and volatile organic compounds are generated during
fermentation by probiotic bacteria. They affect the sensory acceptability
of the product. One of these metabolites, ethanol, gives the beverage a
refreshing taste, while esters give it a fruity odor and provide sensory
qualities that are unique to the product (Fiorda et al., 2017). A total of
21 aroma components, including 10 aldehydes, 6 alcohols, 1 terpene, 2
ketones, 1 ester, and 1 nitrogenous substance, were found in kefir
samples (Table 4). On the 1st, 15th, and 30th days of storage, all kefir
samples included ethanol, ethyl acetate, 2-methyl butanal, 3-methyl
butanal, and hexanal. Solely acetaldehyde, diacetyl, 3-hydroxy-2-buta­
none, and oxime methoxy phenyl compounds were found in kefir
made from cow’s milk, whereas heptanal, octanal, and a-pinene were
found in kefir made from peanut milk, and 1-hexanol was found in kefir
made from walnut milk. In addition, all kefir samples, except cow kefir,
included octenal, nonal, and 1-phenyl butanol (Table 4).
Yeasts are microorganisms that produces alcohol in kefir. Yeasts use
the Embden-Meyerhof-Parnas Path (Glycolytic Pathway) to form
ethanol and carbon dioxide from monosaccharides (Lynch, Wilkinson,
Daenen, & Arendt, 2021). Alcohol enhances the aroma by serving as a
solvent for other olfactory components. The product’s high alcohol
concentration might give it a yeasty flavor (Chen, Lu, Yu, Chen, & Tian,
2019). Ethanol was not detected in hazelnut, peanut, or almond milks;
however, it was detected in trace amounts in cow, cashew, and walnut
milks. Ethanol was the most prevalent volatile observed in kefir samples
after fermentation, with the highest ethanol value (963.86 mg/L) found
in kefir samples made from cow’s milk on the 30th day of storage.
Furthermore, the level of ethanol in kefir samples made from nut milks
during storage increased between the 1st and 15th days of storage and
reduced between the 15th and 30th days of storage (Table 4). When the
environment is low of sugar, yeasts begin to use respiratory substrates
like ethanol as a source of loss for aerobic respiration (Casal, Paiva,

8
E.M. Comak Gocer and E. Koptagel Food Chemistry 402 (2023) 134252

Table 4
Volatile organic compounds (mg/L) of kefir samples during various storage times.
Storage time (days) Cow Cashew Hazelnut Peanut Wallnut Almond

Acetaldehyde 1 1.43 ± 0.03 n.d n.d n.d n.d n.d

15 1.17 ± 0.01 n.d n.d n.d n.d n.d
30 0.88 ± 0.04 n.d n.d n.d n.d n.d
Ethanol 1 147.20 ± 9.03 66.41 ± 5.17 21.44 ± 3.03 33.85 ± 2.31 1.62 ± 0.21 53.33 ± 4.01
15 527.63 ± 13.07 106.76 ± 8.69 53.71 ± 5.11 95.83 ± 2.06 4.35 ± 0.59 63.63 ± 3.43
30 963.87 ± 15.06 92.36 ± 7.42 41.06 ± 3.51 79.33 ± 2.83 1.33 ± 0.04 60.56 ± 2.55
Diacetyl 1 0.21 ± 0.01 n.d n.d n.d n.d n.d
15 0.14 ± 0.04 n.d n.d n.d n.d n.d
30 0.12 ± 0.03 n.d n.d n.d n.d n.d
3-hydroxy-2 butanone 1 0.51 ± 0.02 n.d n.d n.d n.d n.d
15 0.32 ± 0.08 n.d n.d n.d n.d n.d
30 0.31 ± 0.03 n.d n.d n.d n.d n.d
3-methyl-butanol 1 25.75 ± 2.03 n.d n.d n.d n.d n.d
15 15.10 ± 1.31 n.d n.d n.d n.d n.d
30 10.63 ± 1.57 n.d n.d n.d n.d n.d
Oxime-. methoxy-phenyl- 1 21.33 ± 2.48 n.d n.d n.d n.d n.d
15 8.73 ± 1.14 n.d n.d n.d n.d n.d
30 3.16 ± 0.63 n.d n.d n.d n.d n.d
Ethyl acetate 1 17.44 ± 2.25 4.18 ± 0.63 1.96 ± 0.30 2.09 ± 0.08 0.24 ± 0.02 3.68 ± 0.25
15 25.37 ± 2.74 11.29 ± 1.09 4.29 ± 0.18 5.39 ± 0.37 0.38 ± 0.01 6.69 ± 0.62
30 21.84 ± 1.29 9.41 ± 1.01 2.86 ± 0.69 3.53 ± 0.44 0.12 ± 0.01 4.83 ± 0.89
Heptane 1 n.d n.d n.d n.d n.d n.d
15 n.d n.d n.d n.d n.d n.d
30 n.d n.d n.d n.d n.d n.d
2-methyl-butanal 1 0.04 ± 0.01 0.19 ± 0.01 0.10 ± 0.01 0.04 ± 0.01 0.12 ± 0.06 0.44 ± 0.05
15 0.06 ± 0.01 0.21 ± 0.02 0.12 ± 0.03 0.06 ± 0.02 0.17 ± 0.05 0.52 ± 0.12
30 0.03 ± 0.01 0.10 ± 0.02 0.09 ± 0.01 0.04 ± 0.01 0.13 ± 0.02 0.42 ± 0.01
3-methyl-butanal 1 0.07 ± 0.01 0.48 ± 0.12 0.13 ± 0.02 0.03 ± 0.01 0.10 ± 0.01 0.37 ± 0.02
15 0.09 ± 0.04 0.60 ± 0.23 0.17 ± 0.04 0.04 ± 0.02 0.13 ± 0.01 0.47 ± 0.08
30 0.06 ± 0.02 0.38 ± 0.02 0.11 ± 0.01 0.02 ± 0.01 0.09 ± 0.02 0.30 ± 0.01
Pentanal 1 0.03 ± 0.01 0.47 ± 0.01 0.10 ± 0.03 0.02 ± 0.01 0.05 ± 0.03 0.91 ± 0.06
15 0.04 ± 0.01 0.59 ± 0.14 0.14 ± 0.02 n.d 0.04 ± 0.01 1.15 ± 0.07
30 0.03 ± 0.02 0.30 ± 0.02 0.09 ± 0.02 n.d 0.03 ± 0.01 0.75 ± 0.09
Hexanal 1 0.06 ± 0.01 0.98 ± 0.21 0.17 ± 0.05 0.26 ± 0.02 1.62 ± 0.21 0.21 ± 0.08
15 0.12 ± 0.02 0.97 ± 0.17 0.20 ± 0.06 0.32 ± 0.01 2.02 ± 0.27 0.30 ± 0.02
30 0.09 ± 0.01 0.83 ± 0.13 0.13 ± 0.04 0.21 ± 0.03 1.33 ± 0.10 0.20 ± 0.08
Heptanal 1 n.d n.d n.d 0.09 ± 0.02 n.d n.d
15 n.d n.d n.d 0.10 ± 0.03 n.d n.d
30 n.d n.d n.d 0.07 ± 0.02 n.d n.d
Octenal 1 n.d 0.15 ± 0.05 0.07 ± 0.02 0.09 ± 0.03 0.12 ± 0.01 0.19 ± 0.04
15 n.d 0.13 ± 0.01 0.08 ± 0.03 0.10 ± 0.02 0.14 ± 0.02 0.18 ± 0.01
30 n.d 0.09 ± 0.03 0.05 ± 0.01 0.07 ± 0.01 0.08 ± 0.01 0.13 ± 0.04
Octanal 1 n.d n.d n.d 0.15 ± 0.01 n.d n.d
15 n.d n.d n.d 0.11 ± 0.02 n.d n.d
30 n.d n.d n.d 0.08 ± 0.01 n.d n.d
1-hexanol 1 n.d n.d n.d n.d 0.36 ± 0.01 n.d
15 n.d n.d n.d n.d 0.37 ± 0.04 n.d
30 n.d n.d n.d n.d 0.18 ± 0.02 n.d
Nonanal 1 n.d 0.20 ± 0.07 0.33 ± 0.04 0.07 ± 0.01 0.49 ± 0.07 0.68 ± 0.03
15 n.d 0.20 ± 0.01 0.30 ± 0.02 0.08 ± 0.02 0.52 ± 0.06 0.74 ± 0.10
30 n.d 0.13 ± 0.05 0.28 ± 0.01 0.06 ± 0.01 0.34 ± 0.02 0.50 ± 0.06
1-heptanol 1 n.d n.d n.d n.d 0.02 ± 0.01 0.03 ± 0.01
15 n.d n.d n.d n.d 0.03 ± 0.01 0.07 ± 0.02
30 n.d n.d n.d n.d 0.22 ± 0.02 0.35 ± 0.06
Benzaldehyde 1 0.03 ± 0.01 0.99 ± 0.01 0.36 ± 0.05 0.06 ± 0.02 0.70 ± 0.09 1.06 ± 0.27
15 n.d 1.28 ± 0.14 0.50 ± 0.07 0.07 ± 0.01 0.84 ± 0.16 1.31 ± 0.14
30 n.d 0.83 ± 0.07 0.29 ± 0.03 0.05 ± 0.01 0.57 ± 0.05 0.82 ± 0.12
1-Phenyl-1-butanone 1 n.d 0.07 ± 0.01 n.d 0.05 ± 0.02 0.02 ± 0.01 0.05 ± 0.01
15 n.d 0.09 ± 0.01 n.d 0.07 ± 0.02 0.02 ± 0.01 0.07 ± 0.01
30 n.d 0.06 ± 0.03 n.d 0.04 ± 0.01 0.01 ± 0.01 0.04 ± 0.01
Phenyl ethyl alcohol 1 n.d 0.06 ± 0.02 0.02 ± 0.01 0.03 ± 0.01 0.03 ± 0.01 n.d
15 n.d 0.07 ± 0.04 0.03 ± 0.01 0.04 ± 0.02 0.04 ± 0.02 n.d
30 n.d 0.04 ± 0.02 0.02 ± 0.01 0.03 ± 0.01 0.02 ± 0.01 n.d
a-pinene 1 n.d n.d n.d 0.03 ± 0.01 n.d n.d
15 n.d n.d n.d 0.04 ± 0.01 n.d n.d
30 n.d n.d n.d 0.03 ± 0.01 n.d n.d

n.d. not detected.

Queirós, & Soares-Silva, 2008). In our study, the amount of glucose in
kefir samples produced from nut milks decreased during storage. It is
thought that the amount of ethanol decreases as of the 15th day of
storage, since there is not enough sugar left in the environment for the
growth of yeasts.
The esterification of short-chain free acids with alcohol produces
volatile esters (Chen, Lu, Yu, Chen, & Tian, 2019). It is generated by
yeast metabolism and released into the fermentation medium in most
cases, except for substrate-derived esters. Acetate esters are frequent in
fermented beverages (Liszkowska & Berlowska, 2021). In our study, no

9
E.M. Comak Gocer and E. Koptagel
ester was found in milk samples (Table S5). The major ester group
component observed in kefir samples after fermentation was ethyl ace­
tate. The highest ethyl acetate value (25.36 mg/L) was found in the cow
kefir on the 15th day of storage (Table 4). Due to its fruity flavor, ethyl
acetate has a considerable impact on the sensory qualities of fermented
beverages. It is also utilized as a food flavoring ingredient. When the
level of ethyl acetate in the solution exceeds 150 mg/L, the vinegar
flavor becomes noticeable. The increase in ethyl acetate content has
been attributed to bacterial degradation produced by acetic acid bac­
teria during long-term storage (Magalhèes-Guede et al., 2018). After
ethanol, ethyl acetate was found to be the most abundant volatile
organic component in kefir samples made from nut milks in our inves­
tigation. The amount of ethyl acetate found in kefir samples increased
until the 15th day of the storage, after which it declined. While acetal­
dehyde is the most prevalent aldehyde in cow kefir, benzaldehyde is the
most prevalent aldehyde in kefir made from nut milks, with the highest
benzaldehyde value (1.31 mg/L) found in almond kefir on the 15th day
of storage (Table 4). Acetaldehyde is a component of fermented foods
that gives them their distinct odor. At low doses, it promotes fruity
aroma, while at large quantities (>200 mg/kg), it degrades the aroma of
fermented foods. Alcohol dehydrogenase can convert acetaldehyde to
ethanol (Braga et al., 2013). During storage, acetaldehyde reduced while
ethanol increased in kefir made from cow’s milk in our findings. Benz­
aldehyde, a sweet, flowery, and spice-like molecule in the aldehyde
group, is employed as a bitter almond taste in the food industry (Dionisio
et al., 2012). Benzaldehyde was found in trace levels in nut kefirs. 2-
methyl butanal and 3-methyl butanal, found in minimal levels in all
kefir samples, are branched chain aldehydes that give nice smell to the
dairy products. In nut kefirs, the ketone group (diacetyl,3-hydroxy-2-
butanone) and nitrogenous substances (methoxyphenyl oxime) were not
determined. Only the kefir from peanut milk contained the a-pinene
from the terpenes group.
4. Conclusion
The amount of carbohydrate, protein, and fat in milk influences the
metabolism and stability of microorganisms in the kefir starter culture,
as well as substrate consumption and metabolite production. With its
high fat and low carbohydrate content, nut milks present a tough
environment for fermentation when compared to cow’s milk. Nut’s milk
is fermented more slowly than cow’s milk because their carbohydrate
content is lower. As a result, the metabolite concentration of the fer­
mented product is lower, and the pH value is greater. The walnut kefir’s
pH did not drop to the necessary level (pH 4.6) at the end of the incu­
bation, which is likely due to the low carbohydrate content of walnut
milk. In cow’s milk with a high carbohydrate content, on the other hand,
a faster fermentation occurs, resulting in a fermented product with a
high metabolite concentration. Unsaturated fatty acids (particularly
oleic, linoleic, and -linolenic acids) of nut milks and their respective
kefirs were higher than that of cow’s milk and respective kefir. Besides,
saturated fatty acid content of nut’s milk and kefir was low, which is
beneficial for health. Cashew kefir has the highest total organic acid
content. Ethanol and ethyl acetate were dominating volatiles among the
aroma components. They were highest in the cow kefir. Nut kefirs made
without additives and/or sugar are suitable products for vegans and
consumers with health issues such as cow’s milk protein allergy and
lactose intolerance.
Funding
This work was supported by the Scientific Research Projects Coor­
dination Unit of Akdeniz University (Project number: TSA-2020-5316).
CRediT authorship contribution statement
Emine Mine Comak Gocer: Conceptualization, Investigation,
10
Food Chemistry 402 (2023) 134252
Methodology, Project administration, Supervision, Visualization,
Writing – review & editing. Ebru Koptagel: Formal analysis, Investi­
gation, Visualization, Writing – original draft.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
No data was used for the research described in the article.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2022.134252.
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