University of Science and Technology of Southern Philippines

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

PROPAGATION AND EVALUATION OF KEFIR GRAINS

FROM FRESH 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 21, 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 fermenting at 48 h under 20 to
30 °C. The increments in grains’ biomass was 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.

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 a fermented beverage. 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 substrate 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 under 20 to 30 °C. 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 was 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 with a 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

4
the salinity of the sample, a 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 place on the magnetic stirrer with a hot plate (Suntex, SH-
301A) set-up. A laboratory thermometer was attached on 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 a 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.

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RESULTS AND DISCUSSION

6
 CONCLUSION AND RECOMMENDATION

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 APPENDICES

Appendix A.

Table 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 2. Fermented Fresh Milk Filtrate

Materials Weight (g)

Milk + Kefir grain + container 335.51

Milk Filtrate + container 319.42

Kefir 16.09
Appendix C.
 REFERENCES

Garrote, G. L., Abraham, A. G., De Antoni, G. L., Mozzi, F., Raya, R. R., & Vignolo, G. M.

(2010). Biotechnology of Lactic Acid Bacteria: Novel Applications.

Chen, Z., Shi, J., Yang, X., Nan, B., Liu, Y., & Wang, Z. (2015). Chemical and physical

characteristics and antioxidant activities of the exopolysaccharide produced by Tibetan

kefir grains during milk fermentation. International Dairy Journal, 43, 15–21.

https://doi.org/10.1016/j.idairyj.2014.10.004

Prado, M. R., Blandón, L. M., Vandenberghe, L. P. S., Rodrigues, C., Castro, G. R., Thomaz-

Soccol, V., & Soccol, C. R. (2015). Milk kefir: composition, microbial cultures,

biological activities, and related products. Frontiers in Microbiology, 6, 1177.

https://doi.org/10.3389/fmicb.2015.01177

Kesenkaş, H., Gürsoy, O., & Özbaş, H. (2017). Kefir. In J. Frias, C. Martinez-Villaluenga, & E.

Peñas (Eds.), Fermented Foods in Health and Disease Prevention (pp. 339–361).

Elsevier.

Prado, M.R.; Blandón, L.M.; Vandenberghe, L.P.S.; Rodrigues, C.; Castro, G.R.; Thomaz-Soccol,

V.; Soccol, C.R. Milk kefir: Composition, microbial cultures, biological activities, and

related products. Front. Microbiol. 2015, 6, 1–10.

Fiorda, F.A.; de Melo Pereira, G.V.; Thomaz-Soccol, V.; Rakshit, S.K.; Pagnoncelli, M.G.B.;

Vandenberghe, L.P.d.S.; Soccol, C.R. Microbiological, biochemical, and functional

aspects of sugary kefir fermentation—A review. Food Microbiol. 2017, 66, 86–95.

Nielsen, B.; Gürakan, G.C.; Ünlü, G. Kefir: A Multifaceted Fermented Dairy Product. Probiotics

Antimicrob. Proteins 2014, 6,123–135.

Rosa, D.D.; Dias, M.M.S.; Grze´skowiak, Ł.M.; Reis, S.A.; Conceição, L.L.; Peluzio, M.D.C.G.

Milk kefir: Nutritional, microbiological and health benefits. Nutr. Res. Rev. 2017, 30,

82–96.

Hsieh, H.H.; Wang, S.Y.; Chen, T.L.; Huang, Y.L.; Chen, M.J. Effects of cow’s and goat’s milk

as fermentation media on the microbial ecology of sugary kefir grains. Int. J. Food

Microbiol. 2012, 4, 14.

Sarkar, S. Potential of kefir as a dietetic beverage—A review. Br. Food J. 2007, 109, 280–290.