J. Dairy Sci. 98:5974–5982 http://dx.doi.org/10.3168/jds.2015-9697 © American Dairy Science Association®, 2015. Alcohol dehydrogenase activity in Lactococcus chungangensis: Application in cream cheese to moderate alcohol uptake Maytiya Konkit, Woo Jin Choi, and Wonyong Kim1 Department of Microbiology, Chung-Ang University College of Medicine, Seoul 156-756, Republic of Korea ABSTRACT teroides (Molimard and Spinnler, 1996; Bockelmann et al., 1997; Beresford et al., 2001; Bockelmann and Many human gastrointestinal facultative anaerobic Hoppe-Seyler, 2001). Members of the genus Lactococcus and aerobic bacteria possess alcohol dehydrogenase (Schleifer et al., 1985) are used extensively in the dairy (ADH) activity and are therefore capable of oxidizing and food fermentation industry, a multibillion-dollar ethanol to acetaldehyde. However, the ADH activity of biotech industry. Lactococcus lactis has been the major Lactococcus spp., except Lactococcus lactis ssp. lactis, bacteria used in cheese manufacture for over 8,000 yr, has not been widely determined, though they play an with an excess of 1,000 varieties worldwide (Sandine important role as the starter for most cheesemaking and Elliker, 1970). Currently, the genus comprises 9 technologies. Cheese is a functional food recognized species and 4 subspecies with validly published names as an aid to digestion. In the current study, the ADH T (Parte, 2014). activity of Lactococcus chungangensis CAU 28 and 11 Several metabolic properties of Lactococcus serve reference strains from the genus Lactococcus was de- special functions that directly or indirectly affect termined. Only 5 strains, 3 of dairy origin, L. lactis processes, such as flavor development and ripening of ssp. lactis KCTC 3769T, L. lactis ssp. cremoris KCCM T T cheese (Olson, 1990) and the probiotic and prebiotic 40699 , and Lactococcus raffinolactis DSM 20443 , and activity of cheese as a functional food. These functions 2 of nondairy origin, Lactococcus fujiensis NJ317T and T T include depletion of the milk sugar lactose, reduction Lactococcus chungangensis CAU 28 KCTC 13185 , of the redox potential of the cheese, citrate fermenta- showed ADH activity and possessed the ADH gene. All tion, and casein degradation to produce bioactive and these strains were capable of making cheese, but the antimicrobial peptides. These metabolic functions must highest level of ADH activity was found in L. chun- be mediated by enzymes and many reports have been gangensis, with 45.9 nmol/min per gram in tryptic soy published on those that play important roles in cheese, broth and 65.8 nmol/min per gram in cream cheese. especially from L. lactis (Engels et al., 2000; Yvon and The extent that consumption of cheese, following im- Rijnen, 2001). Lactococcus lactis is studied predomi- bibing alcohol, reduced alcohol uptake was observed nantly for its role in fermented dairy products, includ- by following the level of alcohol in the serum of mice. ing fermented milks, sour cream, and soft and hard The results show a potential novel benefit of cheese as cheese (Ward et al., 2002). Lactococcus lactis ssp. lactis a dairy functional food. and L. lactis ssp. cremoris are widely used as starters; Key words: Lactococcus, Lactococcus chungangensis, for example, in Cheddar cheese production where they cream cheese, alcohol dehydrogenase are favored because they are less likely to cause bitter- ness and other flavor defects (Heap, 1998). INTRODUCTION Lactococcus, in particular, has a complex proteolytic system capable of converting milk casein to the free AA Lactic acid bacteria are widely used in many kinds and peptides necessary for growth and acid production. of dairy products in many countries in the world. The The proteolytic system is composed of a proteinase in- lactic acid bacteria group comprises many bacterial volved in the initial cleavage of casein, peptidases that species that play an important role in milk fermenta- hydrolyze the large peptides formed, and transport sys- tion processes, including Lactococcus, Lactobacillus, tems for the uptake of small peptides and AA (Law and Streptococcus thermophilus, and Leuconostoc mesen- Haandrikman, 1997). Many enzymes that are important for degradation of casein and other proteins are present in L. lactis, such as endopeptidase (Muset et al., 1989), aminopeptidase (Thomas and Pritchard, 1987), dipep- Received April 11, 2015. Accepted May 25, 2015. tidase (van Boven et al., 1988), tripeptidase (Bosman 1 Corresponding author: [email protected] et al., 1990), and proline-specific peptidase (Baankreis 5974 QUALITY OF DAIRY PRODUCTS 5975 and Exterkate, 1991). However, not only proteinases ris, and Lactococcus raffinolactis, which are routinely can be found in Lactococcus species but also other cata- used in the dairy industry. bolic enzymes, such as alcohol dehydrogenase. Alcohol dehydrogenase (ADH) metabolizes a wide variety of MATERIALS AND METHODS substrates, including ethanol, retinol, aliphatic alco- hols, hydroxysteroids, and lipid peroxidation products Bacterial Strains (Agarwal, 2001). Many studies have detailed the ADH gene and its activity for Lactococcus lactis ssp. lactis in Lactococcus chungangensis CAU 28T (KCTC 13185T, the field of sugar and ethanol production (Even et al., JCM 17100T, DSM 22330T, CIP 109874T), L. lactis 2001; Palmfeldt et al., 2004; Gaspar et al., 2011; Solem ssp. lactis KCTC 3769T, L. lactis ssp. cremoris KCCM et al., 2013; Costa et al., 2014; Gänzle, 2015). 40699T, L. raffinolactis DSM 20443T, L. lactis ssp. hor- In human health, the effects of ingested alcohol on diniae KCTC 3768T, L. lactis ssp. tructae L105T (DSM different organs, including the brain, depend on the 21502T), Lactococcus garvieae KCTC 3772T, L. piscium ethanol concentration achieved and the duration of ex- HRIA 68T (DSM 6634T), Lactococcus plantarum DSM posure. The main site of ethanol metabolism is the liver, 20686T, Lactococcus taiwanensis 0905C15T (NBRC although some metabolism also occurs in other tissues 109049T), Lactococcus formosensis NBRC 109475T, and and can cause local damage there. The main pathway Lactococcus fujiensis 516T (DSM 27937T) were cultured of ethanol metabolism involves its conversion to acetal- in tryptic soy broth (Becton, Dickinson and Company, dehyde, a reaction that is mediated by ADH (Edenberg, Sparks, MD) as a selective medium consisting of a soy- 2007). Many bacteria and other microorganisms (e.g., bean-CN digest and dextrose (Al-Zoreky and Sandine, yeast) throughout the digestive tract can metabolize 1991) at 30°C for 24 h. Strains were obtained from the alcohol. For example, microorganisms normally found Korean Collection for Type Cultures (KCTC; Taejon, in the mouth oxidize alcohol to acetaldehyde in saliva Korea), the Korean Culture Center of Microorganisms (Salaspuro and Salaspuro, 2004). Many studies have (KCCM; Seoul, Korea), the Deutsche Sammlung von linked cheese consumption and health; for example, it Mikroorganismen und Zellkulturen (DSM; Braunsch- has been shown that cheese contains compounds that weig, Germany), and the National Biological Resource reduce the risk of dental caries in children and has a Center (NBRC; Tokyo, Japan). positive effect on dental health (Harper et al., 1986; Krobicka et al., 1987; Papas et al., 1995). The activity Probing ADH Gene in Lactococcus Species of ADH contributed by lactococci in cheese, especially at the higher levels in L. chungangensis, may increase Genomic DNA was extracted using i-genomic BYF the metabolism of alcohol in the gut and decrease the DNA Extraction mini kit (iNtRON Biotechnology, exposure of internal organs to alcohol. Seoul, South Korea). Primers for the ADH genes were Lactococcus chungangensis CAU 28T was isolated designed using Primer 3 software (http://frodo.wi.mit. from activated sludge and characterized by a polypha- edu/primer3/) based on the complete genome sequence sic approach including phenotypic properties, cellular of L. lactis ssp. lactis Il1403 (GenBank accession num- FA composition, and 16S rRNA gene sequence analyses ber NC_002662) and L. lactis ssp. cremoris UC509 (Cho et al., 2008; Konkit et al., 2014). Lactococcus (GenBank accession number CP_003157). The primer chungangensis CAU28T grow under aerobic or faculta- sequences were as follows: forward = 5c-GGTTTGT- tive anaerobic conditions, catalase and oxidase is nega- GACCCAGGTATGGT-3c, and reverse = 5c-GTTC- tive, and it can produce acid from d-glucose, d-fructose, CAAGCACCACCAACTT-3c. The PCR reaction was d-sucrose, d-mannose, and d-mannitol, cellobiose, and 5 U of Taq DNA polymerase (Beams Biotechnology, starch. Leucine aminopeptidase and β-galactosidase Seongnam, South Korea), 2.0 μL of 10× Taq buffer, 1 are also produced by this strain. Analysis of the tran- μL of dNTP mixture, 1 μL each of forward and reverse scriptome of L. chungangensis revealed higher levels of primer, and 3 μL of genomic DNA as a template in a expression of several key enzymes favorable for cheese- total volume of 20 μL. Amplifications were performed making, in line with the increased metabolic diversity in a TProfessional Thermocycler (Biometra GmbH, found in lactococci of nondairy origin, including an el- Göttingen, Germany) as follows: an initial 95°C for 5 evated expression of ADH compared with conventional min, 30 cycles of denaturation with 95°C 1 min, an- starter cultures (Cho et al., 2008; Konkit et al., 2014). nealing 55°C 1 min, extension 72°C 1 min, and a final The objective of the present study was to evaluate the extension at 72°C 10 min. After