Nisin-Producing Lactococcus lactis Strains Isolated from Human Milk

Abstract

Characterization by partial 16S rRNA gene sequencing, ribotyping, and green fluorescent protein-based nisin bioassay revealed that 6 of 20 human milk samples contained nisin-producing Lactococcus lactis bacteria. This suggests that the history of humans consuming nisin is older than the tradition of consuming fermented milk products.One of the most studied bacteriocins, nisin is naturally produced by Lactococcus lactis. Both nisin variants A and Z, with a difference of an amino acid (1), are approved for use in foodstuffs by food additive legislating bodies in the United States (Food and Drug Administration), the United Kingdom, and the European Union (16). Nisin is employed in the dairy industry to inhibit Clostridium botulinum and Bacillus cereus, due to its capacity to prevent spore germination (7,20). Nisin was first identified in fermented cow milk (13). Since then, it has been isolated from various milk and dairy products (8, 16) as well as from plant material (5, 16) and river water (21). In this study, human milk was screened for bacteria to reveal antibacterial activity caused by nisin producers.Early lactational (within 80 days of birth) human milk samples (n ϭ 20) were collected in southern Finland from healthy first-time deliverers and from mothers with several children. Two milk samples were received within a month from one donor. The donors were requested to collect milk in sterile test tubes with minimal skin contact. The fresh milk was screened within hours of delivery for bacteria with antibacterial properties by the agar diffusion test (17). Samples were spotted on nonselective Luria-Bertani agar (14) overlaid with 100 l (at an optical density at 600 nm of 1.6) of Micrococcus luteus A1 NCIMB86166 (National Collection of Industrial and Marine Bacteria), a strain sensitive to many antibacterial substances. Plates covered with M. luteus were allowed to dry with the lid open before 30-l milk sampling and were incubated overnight at 37°C in an aerobic atmosphere. Seven samples from six different mothers contained strains with strong antibacterial activity. Of the 38 isolated strains, 20 colonies produced a clear inhibition zone around M. luteus and were isolated on plates of M17 agar (Oxoid Ltd., Basingstoke, England) with 0.5% (wt/ vol) glucose (M17G) and grown overnight at 30°C.The 20 isolated inhibitory strains from human milk were characterized by partial 16S rRNA gene sequencing. A region of the 16S rRNA gene was amplified by 29 cycles of PCR (consisting of 30 s at 94°C, 60 s at 55°C, and 90 s at 72°C, with a final 120-s extension step at 72°C) with purified chromosomal DNA (18) from the strains as template and using universal primers pA (5Ј AGA GTT TGA TCC TGG CTC AG 3Ј) and pEЈ (5Ј CCG TCA ATT CCT TTG AGT TT 3Ј) (3). The amplified 900-bp fragments were harvested from low-meltingpoint gel LM-3 (Pronadisa, Madrid, Spain), purified with chloroform-propanol (14) The seven L. lactis strains (LL3, 310, 2410, 3A, 4B, 6A, and 6B) were further characterized by the RiboPrinter and compared to the ribop...

Upper Respiratory Tract Microbial Communities, Acute Otitis Media Pathogens, and Antibiotic Use in Healthy and Sick Children

“…Lactococcus species may play a role in protecting the upper respiratory tract microbial community from colonization by potentially pathogenic species. Lactic acid bacteria have been shown to produce a range of antimicrobial substances, such as organic acids, hydrogen peroxide, fatty acids, and bacteriocins (3,4,45). Alternatively, the negative association of Lactococcus and each of the three AOM pathogens may be indicative of a more general mode of competition, such as modulation of host immune responses.…”

Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR

“…These bacterial groups may also play an important role in the reduction of the incidence and severity of infections in the breast-fed infant. In fact, some of the lactic acid bacteria strains isolated from this biological fluid have the ability to inhibit the growth of a wide spectrum of pathogenic bacteria by competitive exclusion and/or through the production of antimicrobial compounds, such as bacteriocins, organic acids, or hydrogen peroxide (3,21,23).…”

Lactic Acid Bacteria as Source of Functional Ingredients

“…Nisin-producing bacteria can be found in about 30% of human milk samples. This substance may protect mothers from mastitis and infants from toxication by pathogenic skin flora like Staphylococcus aureus [124]. Except from nicin, currently pediocin PA-1/AcH from several Pediococcus strains and enterocin AS-48 from Enterococcus faecalis are used as biopreservatives [119].…”