University of Science and Technology of Southern Philippines

Alubijid | Cagayan de Oro | Claveria | Jasaan | Oroquieta | Panaon

EVALUATION OF KEFIR MILK PRODUCED

FROM FRESH MILK AND COCONUT MILK SUBSTRATE

Presented to the Faculty of the

Department of Chemistry
College of Science and Mathematics
University of Science and Technology of Southern Philippines

In Partial Fulfillment of the requirements in

CHEM 326.1: INDUSTRIAL MICROBIOLOGY LABORATORY

PATRICIA BLANCH ALAMBATIN

HOLLIE MARIE BABIA
JOHN NICHOLSON VUELBAN

BS CHEM 3H1

APRIL 24, 2023

 ABSTRACT

Kefir is a form of fermented dairy product which contains renowned probiotics that
coexist symbiotically with other microorganisms such as bacteria and yeasts. Its grains can be
identified according to the substrate it uses in fermentation processes such as dairy or non-dairy
kefir. To exploit the biological and chemical properties of Kefir grains, the laboratory activity
aims to use fresh cow’s milk and coconut milk as substrates of the dairy culture. The
composition and properties of the fermented substrates were evaluated through measuring its
pH, salinity, dissolved sugar, and proteins present after the fermentation process. The
traditional method of propagating dairy kefir grains was used in the laboratory activity where
the grains were added directly into the fresh milk substrate and fermented for 24-48 hours at
room temperature. The increments in grains’ biomass were strictly monitored through its mass
after it was filtered out from the mixture. Similarly, grains were also added directly into the
coconut milk substrate and evaluated if the requirements of growth had been met. Over-
fermentation of fresh milk and coconut milk resulted in a decrease in the mass of kefir grains.
Moreover, pH reduction and decrease in dissolved sugar content were observed for both kefir
milks produced from the two substrates. Precipitate formation when the milk kefir was added
with lactic and acetic acid were also observed, indicating the presence of milk proteins—casein.
Knowing the effects of different substrates on kefir grains can help to improve the quality and
consistency of the final product, optimize the growth and viability of the kefir grains, and create
new and unique flavors of kefir.

Keywords: kefir grains, fermentation, industrial microbiology, functional drink

Alambatin, P.B., Babia, H.M., Vuelban, J.N.

 INTRODUCTION

Kefir is a form of fermented dairy product by adding bacteria and yeast cultures to milk.
The natural sugars in milk provide food for the microorganisms. This enables them to grow
and produce fermented beverages. The characteristic smell and flavor of kefir are due to the
volatile and nonvolatile compounds generated upon fermentation via lipolysis, glycolysis, and
proteolysis. Kefir grains are a kind of yogurt starter, which are white to yellow – white,
gelatinous, and variable in size (varying from 0.3–3.5 cm in diameter) and are composed by a
microbial symbiotic mixture (Garrote et al., 2010; Chen et al., 2015). Kefir grains contain 83
- 90% of active lactic acid bacteria and 10 - 17% of yeast. The most predominantly found
bacterial species in kefir grains are Lactobacillus kefiranofaciens, Lacticaseibacillus paracasei
(basonym Lactobacillus paracasei), Lactiplantibacillus plantarum (basonym Lactobacillus
plantarum), Lactobacillus acidophilus, and Lactobacillus delbrueckii subsp. bulgaricus.
Furthermore, Saccharomyces cerevisiae, S. unisporus, Candida kefyr, and Kluyveromyces
marxianus ssp. marxianus are the predominant yeast species present in kefir. These grains also
contain vitamins (B2, B12, D, K, and A), minerals, amino acids and enzymes. It has been
shown in several studies to have many health benefits and is reported to possess antibacterial,
antifungal, antioxidant, antidiabetic, antitumor activities in addition to being protective against
gastro-intestinal and ischemic heart diseases.

The kefir grains can be categorized depending on the type of substrate used for
fermentation, which can be dairy or non-dairy kefir. In terms of composition, structure,
microorganisms, and metabolic products during fermentation process, both dairy and non-dairy
kefir do not differ significantly from each other. However, there is a grey line that distinguishes
the two substrates from each other and that is the constitution and prevalence of microbial
diversity of kefir grains and the concentration of end bioproducts may differ depending on the
carbon and energy sources (substrate used) available for grain fermentation (Fiorda et.al. 2016;
Hsieh et.al., 2012). A majority of studies reported that dairy kefir, which used milk as substrate
in fermentation process, accentuated its advantages on consumption than a non-dairy kefir
counterpart (Prado et.al., 2015; Fiorda et.al., 2016; Nielsen et.al., 2014). Nevertheless, both
types of substrates are obtained by inoculating the grains into the substrates at varying ratios
(from 1 to 20% w/v) for 18 to 24 hours at 20 to 30 °C.

The quality of raw milk is crucial for kefir production, like in other fermented milk
products. The raw milk for kefir production must fulfill the following requirements: rich
composition, low bacterial and somatic cell counts, and absence of pathogens or inhibitory
substances like antibiotic and disinfectant residues. Although kefir can be produced with milk
from different breeds (cow, goat, and sheep), full-fat, low-fat, or skimmed cow milk is
preferred for industrial production. Meanwhile, a kefir typically contains 80–90% moisture,
0.2% lipid, 3.0% protein, 6.0% sugar, and 0.7% ash, and approximately 1.0% lactic acid and
alcohol (Sarkar, 2007)

Kefir milk is frequently used as a model system in the laboratory for research on the
microbial ecology of fermented dairy products. It is used by researchers to examine the
development and interactions of the different bacteria that make up the kefir grains. This
involves studying the genetic diversity of the microbes as well as the metabolic pathways and
enzymes involved in the fermentation process. In laboratory reports, kefir milk is often
analyzed for its microbial composition, including the types and abundance of bacteria and yeast
present. This information may provide insight on the kefir milk fermentation process and its
possible health advantages. The microbiological makeup of kefir grains and kefir milk may
also be studied, as well as the effects of various fermentation conditions on the end product. In
addition to tracking pH changes and other physical and chemical changes in the milk during
fermentation, this may entail examining the kinds and amounts of bacteria and yeast that are
present.

The main objective of this laboratory activity is to propagate the milk kefir grains using
locally sourced fresh cow milk and fresh coconut milk as the substrates. Additionally, it aims
to analyze the effect of kefir grains on the fresh milk and coconut milk's composition and
properties after the fermentation process. The determination of its properties and composition
was assessed on its sugar content, pH level, and protein content of the milk kefir filtrate using
organic acids.

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 MATERIALS AND METHODS

A. Materials
The materials and instruments used in the laboratory activity were kitchen strainer,
kitchen scraper, 1- liter plastic containers, kitchen measuring cup, cheese cloth, plastic twine,
100, 250, and 500-mL beakers, 1-mL dropper, filter paper (Whatman, 125 mm), laboratory
thermometer, 250-mL glass graduated cylinder, iron stand with clamp, top loading balance
(A&D, GF-3000), pH meter (Suntex, SP-2300), magnetic stirrer with hot plate (Suntex, SH-
301A), brix refractometer (Atago, 2311), and a salinity meter (PCBFUN).

The substances used in the laboratory activity were Milk Kefir Grains originated from
Russia, Local Fresh Milk (Highland Fresh) and 10% concentrations of acetic and citric acid.
All reagents used in the laboratory activity were of from high grade.

B. Methods

Propagation of Dairy Kefir Grains. This laboratory activity followed the traditional
method of cultivating dairy kefir grains through direct addition of grains to the fresh milk
substrate and fermenting it for a period of time at room temperature. During the preparation
process, the solution containing kefir grains in the packaging was first filtered out using a
kitchen strainer and allowed to sit for a while. The solution was discarded. Next, the empty
container, strainer, kefir grains residue in the strainer, and a cup with fresh milk were weighed
separately using a top loading balance (A&D, GF-3000) and recorded their masses
respectively. After the initial mass of grains and the container were recorded, the grains were
added directly to the container containing one cup of fresh milk. The container was covered
completely with a cheese cloth and was secured tightly with a plastic twine. The mixture was
placed in a dim area at room temperature and was allowed to ferment for 48 hours. Ensuing the
fermentation period, the mixture was strained again discarding the fermented milk while the
grain residue was weighed by indirect weighing to monitor and record the increment of growth
in its biomass using a top loading balance (A&D, GF-3000). The filtered culture, kefir grains,
was inoculated into a fresh substrate with the volume measured as a cup (~237 mL) and was
allowed to grow for the next 48 hours. The procedure was repeated thrice until the desired
growth of grains was achieved. The last milk filtrate was kept for evaluation of its salinity, pH,
brix reading, and protein isolation. The overall process of Dairy Kefir Fermentation is shown
in Figure 1.

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 Meanwhile, the Kefir grains residue was inoculated again into a new substrate,
however, this time, coconut milk was used. First, the initial mass of the kefir was recorded.
Then, the grains were added directly into the new substrate in a 1:1 ratio. The mixture was
fermented for 48 hours at room temperature. Subsequently, the mass of the kefir residue was
recorded, and the fermented coconut milk filtrate was subjected to pH measurement using a
pH meter (Suntex, SP-2300).

Figure 1. Schematic Diagram of Milk Kefir Fermentation Process

Evaluation of Fermented Fresh Milk Filtrate. The filtrate, fermented fresh milk, was
first evaluated through its pH using a pH meter (Suntex, SP-2300). The electrode of the
instrument was submerged into the 100-mL beaker containing ~50-mL filtrate and was allowed
to sit for the reading to stabilize. Next, the filtrate was evaluated through the determination of
the quantity of dissolved sugar and salinity in the sample. Using a brix refractometer (Atago,
2311), the instrument was first calibrated using a standard solution. Then, using a dropper, a
small drop of the sample was introduced onto the refraction prism allowing the sample to
spread completely to the surface. The reading was made through its eyepiece while assuring
enough light passes through the prism. Furthermore, in measuring the salinity of the sample, a

4
salinity meter (PCBFUN) was used. Similar to the brix refractometer, the sample was also
introduced onto its refraction prism. Also, the reading was made through its eyepiece. Repeated
measurements were conducted using the brix refractometer and salinity meter and the average
was only reported. Lastly, the filtrate sample was evaluated through isolating the proteins
present using organic acids. From the sample, 200-mL was obtained and measured using the
glass graduated cylinder and transferred into a 500-mL beaker. The beaker was placed on the
magnetic stirrer with a hot plate (Suntex, SH-301A) set-up. A laboratory thermometer was
attached to the iron stand with clamp set-up to monitor the temperature upon heating. After the
set-up was done, the magnetic stirrer with hot plate was placed into low heat and low stirring.
The sample solution was heated and mixed constantly until it reached 55 °C. After achieving
the temperature, the heat was turned off while constant stirring continued upon the addition of
10% acetic acid. The acid was added slowly into the solution drop by drop using a 1-mL
dropper until precipitation becomes visible. The precipitated sample was filtered using filter
paper (Whatman, 125 mm). After the precipitated protein residue was filtered and dried for a
while, it was weighed indirectly with filter paper using a top loading balance (A&D, GF-3000).
The procedure was repeated with 10% citric acid and compared the yield from the two samples.

5
 RESULTS AND DISCUSSION

The following tables and figures show the results for the activation of milk kefir grains,
fermentation of fresh milk and coconut milk, and separation of milk proteins by precipitation
using organic acids. In the activation of milk kefir grains by addition of fresh milk, the results
showed that there is an increase in the mass of the kefir grains after successive additions of
fresh milk. In the fermentation of coconut milk to kefir grains, a decrease in the mass of the
kefir grains and curd-whey formation was observed. Lastly, the formation of precipitate after
adding organic acids—acetic acid and citric acid—to the milk kefir filtrate indicates the
presence of the milk proteins.

Table 1. Activation of Milk Kefir Grains (Addition of fresh milk)

Day Condition Fresh Milk Wt. of milk Wt. of milk Observations on

added (g) kefir grains kefir grains the milk after
(initial, g) (final, g) fermentation

1 101.31 9.29 13.75 Thick, sour smell

2 104.19 13.75 18.24 Thick, sour smell

3 Room 198.07 18.24 129.9 Thick, less sour

temperature smell

4 211.77 129.9 -- --

5 319.42 -- 16.09 Thick, no smell

Table 1 shows the results in the activation of milk kefir grains at room temperature by
addition of fresh milk. The four days (day 1, 2, 3, and 5) successive additions of fresh milk
have the same observations except that no smell was observed on day 5. There was no weight
and observations recorded on day 4 because the consecutive addition of fresh milk was
interrupted. 48 hours after the first addition of ½ cup of fresh milk to the kefir grains, a
thickening of milk and a sour smell was observed. These observations were also observed after
the addition of ½ cup of fresh milk after 24 hours. The thickening of milk started to double so
the amount of fresh milk added also doubled. On day 3 and 4 of activation, 1 cup of milk was
added, and more thickening of milk and less sour smell was observed on day 3. The thickening

6
of milk that was observed means that the kefir grains are already activated and were effectively
fermenting the fresh milk. The aroma of milk kefir started to improve on day 5 of fresh milk
addition which is also a sign of fermentation of the substrate.

Growth of Milk Kefir Grains

140

120
Mass of milk kefir grains (g)

100

80

60

40

20

0
0 1 2 3 4 5 6
Time (days)

Figure 1. Growth of milk kefir grains during the process of activation

In addition, there was an increase in the weight of milk kefir grains—for the first three
days of activation. This result was expected since after successive fermentations, the biomass
of kefir grains increases slowly, while the bacteria/yeast ratio is steadily maintained in the
matrix (Ertekin-Filiz et al. 2011). The growth of milk kefir grains during the 5-days activation
period is illustrated in figure 1. Kefir grains sometimes need time to adjust and mature before
they begin to multiply. The increase in the mass of the kefir grains that was observed depicts
that the kefir grains already have adjusted to their new environment, which is the fresh milk as
the substrate. However, an abrupt increase in the mass of kefir grains after the addition of fresh
milk in day 3 was observed. This outcome resulted from an incomplete filtration of the milk
sample, which is statistically considered as a gross error. Moreover, there was a decrease in the
mass of the kefir granules on day 5. During the activation process, the consecutive addition and
change of fresh milk was interrupted; over-fermentation may have caused in the decrease of
the kefir grains’ mass.

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Table 2. Evaluation of kefir milk produced from fermented fresh milk and coconut milk

Parameters Fermented fresh milk Fermented coconut milk

Wt. of milk kefir grains (g) 16.09 11.54

Temperature (°C) 24.1 23.8

pH 4.52 4.62

Sugar (°Bx) 8.04 11.80

Salinity below 0.1% below 0.1%

Observation curd and whey were formed curd and whey were formed

The results in the evaluation of fermented fresh milk and coconut milk using the
activated milk kefir grains are shown in table 2. The optimum fermentation time for milk using
kefir grains can vary depending on a few factors, such as the temperature, the amount of milk
and the activity level of the kefir grains. In general, a fermentation time of 24-48 hours is
recommended for milk kefir, with a temperature range of 68-78°F (20-25°C) being ideal. The
fermentation of fresh milk and coconut milk exceeded the ideal fermentation time. Kefir grains
need a specific balance of bacteria and yeast to remain healthy. If the fermentation process goes
on for too long, it can upset this balance, leading to a decrease in weight (Miguel et al. 2013).
The decrease in the mass of kefir grains may have resulted from over-fermentation. Fresh milk
from cow and raw coconut milk are low-acid liquid foods which typically have a pH of around
6.6 and 6.2, respectively. At 24.1 °C, the observed pH of fermented fresh milk is 4.52.
Meanwhile, the pH of fermented coconut milk is 4.62 at 23.8 °C. Typical kefir fermentation
occurs at temperatures between 20 and 25 °C for approximately 24 hours, with pH varying
between 4.2 and 4.6, depending on factors such as temperature, fermentation time, and the
composition of the milk used (Barclay et al. 2001). The lactose from milk (substrate) is degraded
to acid during the fermentation process, which causes pH reduction, increase in consistency,
and sour taste (D'Incecco, P., et al. 2015). Using a brix refractometer (Atago, 2311), the dissolved
sugar content of the filtrate was determined. Compared to the fermented fresh milk, fermented
coconut milk has greater sugar content. The sugar content of milk kefir can vary depending on
several factors, including the type of milk used and the fermentation time. During the
fermentation process, the bacteria and yeasts in the kefir culture consume lactose (a type of
sugar) in the milk, converting it into lactic acid and other compounds. This means that milk

8
kefir typically has a lower sugar content than the milk it was made from. The salinity of the
filtrate was also analyzed using salinity refractometer. The reading for both fermented milks
were below 0.1%. In general, kefir milk does not contain added salt. The lactic acid produced
during fermentation causes the formation of curd and whey which was observed in both the
kefir milk produced from fresh milk and coconut milk.

Table 3. Milk kefir filtrate + fresh milk

Parameters Recorded Values

pH 6.19
Temperature (°C) 25.0

Sugar (°Bx) 7.50

Salinity --

Before performing the separation of milk proteins, the milk kefir filtrate was evaluated
with the same parameters. Table 4 shows the results in the evaluation of pure milk kefir filtrate
when added with fresh milk. At 25.0 °C, the pH of the milk kefir filtrate one week after the
addition of fresh milk is 6.19. The pH increased since the fresh milk was added with pure milk
kefir filtrate, there were no kefir grains added that would cause a decrease of pH of the milk.
The value of the sugar content is 7.50 °Bx.

Table 4. Separation of milk protein by precipitation using organic acids

Type of organic acid used 10% Acetic acid 10% Citric acid

Weight of precipitate, (g) 54.65 50.48

Percent yield of casein, (%) 25.7 24.6

Observation Cloudy, a precipitate was Cloudy, a precipitate was
formed formed

Table 4 illustrates the results in the separation of milk protein by precipitation using
organic acids—acetic acid and citric acid. Two 200 mL milk samples from milk kefir filtrate

9
were evaluated by slowly adding 10% acetic and 10% citric acid in each milk sample until
formation of precipitate is complete. The observation of cloudy solution indicates precipitate
formation. The formation of precipitate due to the neutralization of casein micelles (when
added with acid) suggests the presence and separation of the milk protein—casein. While kefir
is known to contain a variety of beneficial microorganisms, it does not typically contain a high
amount of protein. The protein content of milk kefir grains themselves is relatively low, with
most of the protein in kefir coming from the milk used to make it (Cagindi & Ot1es, 2003).
The exact protein content of milk kefir grains may vary depending on a variety of factors,
including the type of milk used, the fermentation conditions, and the age and health of the kefir
grains themselves (Cagindi & Ot1es, 2003). However, in general, milk kefir grains are not
considered a significant source of protein. In addition, milk kefir also contains organic acids
e.g., lactic acid, acetic acid, and citric acid. The concentrations of organic acids in fermented
milk mainly vary with the type of milk, the type and composition of the starter culture and the
duration of fermentation (Arifoglu et al. 2014).

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 CONCLUSION AND RECOMMENDATION

The purpose of this laboratory experiment is to determine the effects of different

substrates—fresh milk and coconut milk—on the biomass of kefir grains. The composition and
properties of the fermented substrates were evaluated through measuring its pH, salinity,
dissolved sugar, and proteins present after the fermentation process. The experiment revealed
that there was an increase in the mass of kefir grains when added to the fresh milk substrate.
On the other hand, over-fermentation of fresh milk and coconut milk resulted in a decrease in
the mass of kefir grains. Moreover, pH reduction and decrease in dissolved sugar content were
observed. Precipitate formation when the milk kefir was added with lactic and acetic acid were
also observed, indicating the presence of milk proteins—casein. Kefir is a natural functional
dairy product, and its consumption is important. Understanding the effects of different
substrates on kefir grains is important for several reasons. First, it can help to optimize the
fermentation process and ensure consistent quality and flavor in the final product. Second, it
can help to identify which substrates are most compatible with the specific strains of bacteria
and yeast in the kefir grains, which can improve their growth and viability. Finally, it can help
to create new and unique flavors of kefir by experimenting with different substrates, which can
be beneficial for commercial producers looking to differentiate their products in a crowded
market.

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 APPENDICES

Appendix A.

Table A.1: Fermentation of Kefir

Materials Weight (g)

Container 15.61

Container + Strainer 24.65

Kefir 9.29

Measuring cup 15.41

Measuring cup + Milk 101.31

Kefir + Container + Milk 126.00

Appendix B.

Table B.2. Fermented Fresh Milk Filtrate

Materials Weight (g)

Milk + Kefir grain + container 335.51

Milk Filtrate + container 319.42

Kefir 16.09
Appendix C.

Table C.3. Separation of milk protein by precipitation using organic acids

Type of organic acid used 10% Acetic acid 10% Citric acid

Weight of filter paper, (g) 1.6545 1.6671

Weight of filter paper + 56.30 52.15

precipitate, (g)

Mass of 200 mL milk:

1.025 g/cm3 x 200 cm3 = 205 g of milk

% yield of casein:

𝑚𝑎𝑠𝑠 𝑜𝑓 𝑐𝑎𝑠𝑒𝑖𝑛 (𝑝𝑟𝑒𝑐𝑖𝑝𝑖𝑡𝑎𝑡𝑒)

% yield = 𝑥 100
𝑚𝑎𝑠𝑠 𝑜𝑓 𝑚𝑖𝑘