J. Dairy Sci.

99:4999–5007
http://dx.doi.org/10.3168/jds.2016-11002
© American Dairy Science Association®, 2016.

Activities of amylase, proteinase, and lipase enzymes from Lactococcus

chungangensis and its application in dairy products
Maytiya Konkit and Wonyong Kim1
Department of Microbiology, Chung-Ang University College of Medicine, Seoul 06974, Republic of Korea

ABSTRACT ucts, and ensure consistency of the process and product

quality. Lactococcus is a genus of LAB that serves as a
Several enzymes are involved in the process of convert- starter for several types of cheese, such as hard cheese
ing milk to lactic acid and coagulated milk to curd and, without eyes or with small eyes (Leroy and De Vuyst,
therefore, are important in dairy fermented products. 2004), Cheddar (Cretenet et al., 2011), Moroccan soft
Amylase, proteinase, and lipase are enzymes that play white cheese (Ouadghiri et al., 2005), Domiati cheese
an important role in degrading milk into monomeric (El-Baradei et al., 2007), and cream cheese (Konkit et
molecules such as oligosaccharides, amino acids, and al., 2015).
fatty acids, which are the main molecules responsible Lactococcus lactis ssp. lactis, Lactococcus lactis ssp.
for flavors in cheese. In the current study, we deter- cremoris, and Lactococcus raffinolactis are well-known
mined the amylase, proteinase, and lipase activities of strains used as starters in cheese and fermented milk
Lactococcus chungangensis CAU 28T, a bacterial strain (Leroy and De Vuyst, 2004; Ouadghiri et al., 2005). For
of nondairy origin, and compared them with those of many fermented dairy products, the most important
the reference strain, Lactococcus lactis ssp. lactis KCTC properties and functions of LAB are (1) fermentation,
3769T, which is commonly used in the dairy industry. including the depletion of milk sugar and the production
Lactococcus chungangensis CAU 28T and L. lactis ssp. of acids in milk; (2) reduction of the redox potential;
lactis KCTC 3769T were both found to have amylase, (3) citrate fermentation; and (4) casein degradation
proteinase, and lipase activities in broth culture, cream (Olson, 1990). The primary role of LAB, however, is
cheese, and yogurt. Notably, the proteinase and lipase to utilize milk as a substrate and convert it into mono-
activities of L. chungangensis CAU 28T were higher meric molecules for use as nutrients for their metabo-
than those of L. lactis ssp. lactis KCTC 3769T, with lism and growth (Vihinen and Mantsiila, 1989). These
proteinase activity of 10.50 U/mL in tryptic soy broth changes during the fermentation process, including the
and 8.64 U/mL in cream cheese, and lipase activity secretion of nutritional and chemical substances, are
of 100 U/mL of tryptic soy broth, and 100 U/mL of associated with bacterial enzymes (Gurr, 1987).
cream cheese. In contrast, the amylase activity was low, Microbial enzymes are more valuable in manufactur-
with 5.28 U/mL in tryptic soy broth and 8.86 U/mL in ing than animal and plant enzymes because of their va-
cream cheese. These enzyme activities in L. chungan- riety of catalytic activities and the high yields possible
gensis CAU 28T suggest that this strain has potential (Seitz, 1990; Kunji et al., 1995). Furthermore, their
to be used for manufacturing dairy fermented products, rapid growth on inexpensive media and the stability
even though the strain is of nondairy origin. of their enzyme products make bacteria the preferred
Key words: Lactococcus chungangensis, amylase, enzyme source in the food industry (McSweeney and
proteinase, lipase, dairy product Sousa, 2000). Among the technical enzymes, amylase,
proteinases, and lipases are the principal enzymes used
INTRODUCTION in food and animal feed production. Amylase catalyzes
the hydrolysis of starch, resulting in products such as
Lactic acid bacteria (LAB) have been widely used glucose, maltose, and maltotriose units (Gupta et al.,
in the dairy industry to produce cheese, yogurt, butter, 2003; Kandra, 2003; Rajagopalan and Krishnan, 2008).
and fermented milk. The LAB often play an important Amylase was the one of the first indigenous enzymes to
role in the starter cultures of commercial dairy prod- be identified in milk, with α-amylase being the princi-
pal enzyme and β-amylase the lesser (Sato, 1920). Milk
starch is broken down by amylase and converted into
Received February 8, 2016.
Accepted March 4, 2016. primarily dextrin and then into maltose, to a lesser
1
Corresponding author: [email protected] extent (Guzmán-Maldonado et al., 1995).

4999
5000 KONKIT AND KIM

The proteolytic systems of LAB are essential for their MATERIALS AND METHODS
growth in milk and contribute significantly to the de-
velopment of flavor in fermented milk products (Kunji Bacteria Strains
et al., 1995, 1996). Proteinase is the one of the enzymes
Lactococcus chungangensis CAU 28T and L. lactis ssp.
that converts milk casein to free AA and peptides
lactis KCTC 3769T were cultured in tryptic soy broth
necessary for growth and acid production (Law and
(Becton, Dickinson and Co., Sparks, MD) at 30°C for
Haandrikman, 1997). Some Lactococcus strains serve
24 h. Lactococcus lactis ssp. lactis KCTC 3769T was
as starters for dairy fermentation and have proteolytic
obtained from the Korean Collection for Type Cultures
activity. Proteolysis is the first biochemical step in the
(KCTC; Taejon, Korea).
process that determines the flavor and texture of dairy
products (Fox et al., 1996; McSweeney and Fox, 1997).
Specifically, lactococci possess a proteolytic system Broth Culture with Each Enzyme Substrate
that, together with other protein-hydrolyzing enzymes,
is responsible for the conversion of casein into peptides One percent (wt/vol) each of starch, casein, and
and AA. olive oil was added to basal medium, as described by
Lipolytic enzymes are involved in the breakdown Zhang et al. (1983), which contained (g/L) (NH4)2SO4
and mobilization of lipids within the cells of an indi- 1.0, K2HPO4 6.0, KH2PO4 3.0, MgSO4·7H2O 0.01,
vidual organism as well as the transfer of lipids from CaCl2·2H2O 0.05, MnSO4·2H2O 0.01, FeSO4·7H2O
one organism to another (Beisson et al., 2000). Milk 0.001, and ZnSO4·7H2O 0.001. An inoculum of each
fat is essential for the development of a desirable flavor strain was added (1.0%, vol/vol) to each broth culture
in fermented dairy products. Lipase is the enzyme that and then incubated at 30°C for 54 h.
hydrolyzes triglycerides to fatty acids and glycerol,
and mono- or diglycerides that are the key to flavor Cream Cheese Making
development (McSweeney and Sousa, 2000). Bacillus
species have been found to possess amylase, protein- According to a published method used for making
ase, and lipase enzymes that can be used in the food cream cheese (Konkit et al., 2015), pasteurized milk
and household industries (Hasan et al., 2006). Among (Pasteur Milk Co. Ltd., Seoul, Korea) was heated at
those, Bacillus subtilis is a well-known enzyme-produc- 68°C for 30 min and cooled down, 5% (vol/vol) of a
ing species that plays an important role in the produc- starter Lactococcus strain was added, and the mixture
tion of natto by solid-state fermentation of soybeans was incubated at 30°C for 48 h. During this period, the
(Hara and Ueda, 1982). Their proteinase enzymes have milk was acidified. It was then stirred and heated at
been used in the production of household and heavy 70°C for 5 min, and subsequently the whey was sepa-
detergents (Schallmey et al., 2004). Moreover, Bacillus rated through a cloth bag. The curd was set and whey
species have been used as probiotics (Ziaei-Nejad et drained by adding 0.5% salt. Finally, each cream cheese
al., 2006). sample was freeze-dried and stored in the dark at 4°C
Lactococcus chungangensis CAU 28T is a strain of non- until further tests.
dairy origin, which was isolated from activated sludge
in our laboratory (Cho et al., 2008). Transcriptomic Yogurt Making
analysis showed that the strain possessed genes such as
cystathionine β-lyase (MetC) and O-acetylserine sulf- Pasteurized milk was heated to about 93°C and
hydrylase (CysK), which are important factors in the stirred gently to prevent it from boiling over and then
processing of cheese (Konkit et al., 2014). Moreover, cooled to around 44 to 46°C. The milk was stirred oc-
the strain has been found to play a role in functional casionally to prevent skin formation; then, a cup of
activities, such as alcohol dehydrogenase and aldehyde warm milk was added to the inoculum strain (1% vol/
dehydrogenase, which can moderate the level of alcohol vol) and allowed to set overnight.
and aldehyde in vivo (Konkit et al., 2015, 2016). The
objective of this study was to evaluate the potential Amylase Activity
amylase, proteinase, and lipase activities of L. chun-
gangensis CAU 28T in broth culture, yogurt, and cream Maltose (0.5 M) dilutions ranging from 0.3 to 0.5
cheese. A comparison was made with another culture, μmol/mL, including a blank tube, were prepared. One
L. lactis ssp. lactis KCTC 3769T, which is routinely milliliter of each dilution of maltose was pipetted into
used in the dairy industry. a series of corresponding numbered tubes, and 1 mL

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 ENZYMES USED IN DAIRY PRODUCTS 5001

of dinitrosalicylic acid color reagent was added to each RESULTS

tube. After this, the mixture was incubated in boiling
water for 5 min, cooled to room temperature, and then Cream Cheese Making
10 mL of distilled water was added to each tube and
mixed well. Results (absorbance) were read at 540 nm. Cream cheese was made using L. chungangensis CAU
One milliliter of starch was pipetted into a test tube, 28T and L. lactis ssp. lactis KCTC 3769T as starter cul-
along with 1.0 mL of enzyme solution, and incubated at tures. The products were white and had slightly lactic
25°C for 3 min. Subsequently, 1 mL of dinitrosalicylic acid and cultured diacetyl aromas, with off-flavors. The
acid color reagent was added to each tube. All tubes texture of the products was smooth without lumps or
were incubated in a boiling water bath for 15 min. The grittiness, and there was no cracking or wheying off.
mixtures were cooled to room temperature and 9 mL
of distilled water added. Samples were mixed well and Yogurt Making
read at 540 nm, with values for blank wells subtracted
from the results. Maltose released (μmol) was deter- Yogurt was made using L. chungangensis CAU 28T
mined from the standard curve (Hagberg, 1960). and L. lactis ssp. lactis KCTC 3769T as starter cultures.
Each product was a cream color and had high ten-
sion and firmness with a sour smell. The texture of the
Proteinase Activity product was soft and semi-liquid, and the pH of the
yogurt decreased to 4.6 in 7 d.
Tyrosine (1.1 mM) dilutions ranging from 0.1 to 0.5
μmol/mL, including a blank tube, were prepared. Two
Amylase Activity
milliliters of each dilution of tyrosine was pipetted into
a series of corresponding numbered tubes, and 5 mL of In broth culture, basal medium, which was unsupple-
Na2CO3 and 1 mL of Folin-Ciocalteu phenol reagent mented, was added to starch. Lactococcus chungangen-
were added to each tube. After this, the mixture was sis CAU 28T was found to have the highest amylase
swirled and incubated at 37°C for 30 min, and then activity, 5.28 U/mL, at 6 h. This level was similar to
filtered using a 0.45-μm syringe filter. Results were that seen with L. lactis ssp. lactis KCTC 3769T, 5.30
read at 660 nm, with blank values subtracted. Five U/mL (Figure 1A and 1B). In addition, in cream cheese
milliliters of casein was pipetted into a test tube, along made with each strain, L. chungangensis CAU 28T had
with 1.0 mL of enzyme solution, and incubated at 37°C the highest activity on d 5 of incubation with 8.86 U/
for 5 min. Following this, 5 mL of trichloroacetic acid mL, whereas L. lactis ssp. lactis KCTC 3769T had the
reagent was added to each tube. All tubes were incu- highest activity on d 7 at 9.03 U/mL (Figure 2A). In
bated at 37°C for 30 min, and then each solution was yogurt, L. chungangensis CAU 28T had the highest level
filtered using a 0.45-μm syringe filter. Two milliliters of amylase activity on d 3 of incubation at 5.41 U/mL
of test filtrate, 5.0 mL of Na2CO3, and 1 mL of 0.5 and a slight decrease from d 5 to 7, whereas L. lactis
M Folin-Ciocalteu phenol reagent were added to each ssp. lactis KCTC 3769T had the highest amylase activ-
tube. The solutions were mixed well and incubated at ity on d 5 at 5.33 U/mL (Figure 2B).
37°C. Samples were then read at 660 nm with blank
values subtracted (McDonald and Chen, 1965).
Proteinase Activity

Lipase Activity Casein was added to each culture broth to estimate

the proteinase activity of L. chungangensis CAU 28T
Three milliliters of olive oil and 1.0 mL of 200 mM and L. lactis ssp. lactis KCTC 3769T. Lactococcus
Tris-HCl were added to a test tube and incubated at chungangensis CAU 28T was found to have the high-
37°C for 10 min. After this, 1.0 mL of enzyme solution est proteinase activity, with 10.50 U/mL at 30 h. This
was added, and the mixture was incubated at 37°C for result was the similar to that of the L. lactis ssp. lactis
30 min, followed by the addition of 3.0 mL of 95% KCTC 3769T culture that showed proteinase activity
ethanol. The solution was mixed well, and then 4 drops of 9.80 U/mL at 30 h (Figure 1A and 1B). In cream
of thymolphthalein was added to each sample and cheese made with each strain, L. chungangensis CAU
to blank test tubes, followed by titration with 0.2 M 28T had the highest proteinase level on d 7 with 0.54
NaOH to determine the activity levels (Stoytcheva et U/mL, whereas L. lactis ssp. lactis KCTC 3769T had
al., 2012). the highest level on d 7 with 0.41 U/mL (Figure 3A).

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5002 KONKIT AND KIM

Figure 1. Amylase, proteinase, and lipase activities (U/mL) of Lactococcus chungangensis CAU 28T and Lactococcus lactis ssp. lactis KCTC
3769T in broth culture. Amylase and proteinase activities of (A) L. chungangensis CAU 28T in culture broth; (B) amylase and proteinase activi-
ties of L. lactis ssp. lactis KCTC 3769T in culture broth; and (C) lipase activities in culture broth. Error bars represent SD.

Journal of Dairy Science Vol. 99 No. 7, 2016

 ENZYMES USED IN DAIRY PRODUCTS 5003

In yogurt, L. chungangensis CAU 28T had the highest the same time point (Figure 1C). In cream cheese made
level of proteinase on d 3 with 8.64 U/mL. In contrast, from each strain, L. chungangensis CAU 28T had the
L. lactis ssp. lactis had the highest level of proteinase highest lipase activity with 100 U/mL on d 7 (Figure
on d 7 with 1.09 U/mL (Figure 3B). 4A). This lipase activity was the same as that seen in the
L. lactis ssp. lactis KCTC 3769T culture, which showed
Lipase Activity of L. chungangensis lipase activity of 80 U/mL. In yogurt, L. chungangensis
and L. lactis ssp. lactis CAU 28T had the highest level of lipase activity on d 7
at 100 U/mL, whereas L. lactis ssp. lactis KCTC 3769T
Olive oil was added to each culture broth to estimate showed lipase activity of around 80 U/mL (Figure 4B).
the lipase activity of L. chungangensis CAU 28T and
L. lactis ssp. lactis KCTC 3769T. Lactococcus chungan- DISCUSSION
gensis CAU 28T was found to have the highest lipase
activity, with 100 U/mL at 48 h, whereas L. lactis ssp. For many fermented foods, but particularly milk-
lactis KCTC 3769T had lipase activity of 80 U/mL at derived products, the characterization of microorgan-

Figure 2. Amylase activity (U/mL) of Lactococcus chungangensis CAU 28T and Lactococcus lactis ssp. lactis KCTC 3769T in cream cheese
and yogurt. Amylase activities in (A) cream cheese, and (B) yogurt. Error bars represent SD.

Journal of Dairy Science Vol. 99 No. 7, 2016

5004 KONKIT AND KIM

isms responsible for fermentation led to the isolation They contribute to the taste and texture of fermented
of starter cultures that could be produced on a large products and inhibit food spoilage caused by other
scale for the manufacture of these products (Caplice bacteria (Wouters et al., 2002) by producing growth-
and Fitzgerald, 1999). Starter cultures are important inhibiting substances and large amounts of lactic acid.
to dairy manufacturing because they facilitate rapid As productive agents in fermentation, LAB are involved
acidification (Wouters et al., 2002). in making yogurt, cheese, cultured butter, sour cream,
The LAB are typically involved in the fermentation and sausage (Todar, 2006).
of food and dairy products; LAB belong primarily to In fermented dairy products, LAB are naturally pres-
the order Lactobacillales and are included in genera ent in milk and produce lactic acid, which is required for
commonly used in fermented food production, such as the coagulation of milk. Lactococci are applied in dairy
Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, fermentation. These bacteria acidify the milk, and,
and Streptococcus (Todar, 2006). These are important consequently, the growth of other bacteria is largely
groups of microorganisms used in food fermentation. inhibited. This process has been used for centuries to

Figure 3. Proteinase activity (U/mL) of Lactococcus chungangensis CAU 28T and Lactococcus lactis ssp. lactis KCTC 3769T in cream cheese
and yogurt. Proteinase activities in (A) cream cheese, and (B) yogurt. Error bars represent SD.

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 ENZYMES USED IN DAIRY PRODUCTS 5005

Figure 4. Lipase activity (U/mL) of Lactococcus chungangensis CAU 28T and Lactococcus lactis ssp. lactis KCTC 3769T in cream cheese and
yogurt. Lipase activities in (A) cream cheese, and (B) yogurt.

manufacture fermented dairy products (Wouters et al., have been extensively studied for their biochemical and
2002). physiological characteristics and their effects on food
Starter cultures of mesophilic lactococci are used (Teuber, 1995).
in the manufacture of a broad array of cheese types, The most important properties of microorganisms as
butters, and various fermented milk products. Lac- milk starters are their ability to produce acid in milk
tococci have been used for lactic acid fermentation, and to convert protein into flavor components (Wouters
casein breakdown, diacetyl production from citrate, et al., 2002). Enzyme activity is critical to this process.
and resistance to phage attack. The subspecies of Lac- By the early 20th century, many enzymes serving this
tococcus lactis are of great economic importance and function had been identified in milk, including lactoper-

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5006 KONKIT AND KIM

oxidase, catalase, xanthine oxidase, proteinase, lipase, gensis CAU 28T had the highest lipase activity in all
salolase (arylesterase), and amylase (Fox and Kelly, conditions, showing the highest levels of activity (100
2006). U/mL) in the culture broth with olive oil and in yogurt.
The principle underlying the oxidation of carbohy- Our results show that L. chungangensis CAU 28T has
drates and its derivatives is the generation of end prod- amylase, proteinase, and lipase enzyme activity; few
ucts, which are generally acids, alcohol, and carbon di- studies have determined activity levels of these enzymes
oxide. These end products control the growth of spoilage in LAB or related bacteria. Amylase, proteinase, and
microorganisms. Amylase is an enzyme involved in milk lipase have the ability to degrade substrates in broth
production. Milk contains no starch and only low levels culture, cream cheese, and yogurt. The amylase enzyme
of oligosaccharides; however, these oligosaccharides are activity of this strain was not different compared with
derived from lactose and contain unusual monosaccha- L. lactic ssp. lactis, a strain commonly used in the
rides (e.g., fructose and N-acetylneuraminic acid) with manufacture of dairy products. Interestingly, however,
glycosidic linkages. Therefore, it seems that α-amylase the proteinase and lipase activities of L. chungangensis
is highly specific for α(1→4) glycosidic bonds linking CAU 28T were higher than those of L. lactic ssp. lactis.
glucose molecules in the hydrolysis of oligosaccharides In conclusion, L. chungangensis CAU 28T has amylase,
in milk (Gnoth et al., 2000). proteinase, and lipase activities that are essential to
In this study, L. chungangensis CAU 28T was found to hydrolyzing substrates in milk to lactic acid, which is
have amylase activity in culture broths of starch, cream important in dairy milk fermentation.
cheese, and yogurt. It had the highest level of activity
in cream cheese, with 8.86 U/mL, whereas L. lactis ssp.
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Journal of Dairy Science Vol. 99 No. 7, 2016