Towards application of water extract from heat-killed Lactococcus lactis H61 as a cosmetic ingredient.

We previously reported that oral administration of heat-killed Lactococcus lactis H61 improves certain human skin properties. For topical application of this strain, we reasoned that a bacterial cell extract obtained with an aqueous solvent could be readily formulated as a cosmetic ingredient. In the present study, we characterized the water extract from heat-killed H61. The extract had inhibitory activity for angiotensin-converting enzyme, which is known as suppression of inflammation of skin, and absorbed electromagnetic radiation in the UVB range. UVB-irradiated normal human epidermal keratinocytes (NHEKs) had lower viability than nonirradiated NHEKs. The NHEK survival rate was significantly higher in cells treated with the extract at 10 mg dried cells per ml prior to UVB exposure than in untreated cells or cells treated with lower extract concentrations. At this concentration, the extract also inhibited the production of interleukin-8 induced by UVB. The extract did not protect against hydrogen peroxide-induced cell damage. These data indicate that topical application of the H61 extract alleviates UVB damage and reduces inflammation in skin cells. The present study expands the potential application of strain H61 to its use as a cosmetic ingredient in addition to its use in the food industry. SIGNIFICANCE AND IMPACT OF THE STUDY: In our previous report, oral administration of heat-killed Lactococcus lactis H61 improved certain human skin properties. This study aimed exploring the potential topical use of this strain. The water extract derived from heat-killed cells with angiotensin-converting enzyme inhibitory activity, which is known as suppression of inflammation of skin, could protect normal human epidermal keratinocytes (NHEKs) from damage caused by UVB. Higher interleukin-8 production by UVB-exposed NHEKs than nontreated cells was suppressed by addition of the extract. The extract absorbed electromagnetic radiation in the UVB range. This extract could help in the maintenance of skin health by suppressing inflammation.

Effects of rare sugar D-allulose on acid production and probiotic activities of dairy lactic acid bacteria.

It has recently been reported that the rare sugar d-allulose has beneficial effects, including the suppression of postprandial blood glucose elevation in humans, and can be substituted for sucrose as a low-calorie food ingredient. To examine the applications of d-allulose in the dairy industry, we investigated the effects of d-allulose on the acid production of 8 strains of yogurt starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and 4 strains of lactococci, including potential probiotic candidates derived from dairy products. Acid production by 2 L. delbrueckii ssp. bulgaricus yogurt starter strains in milk was suppressed by d-allulose, but this phenomenon was also observed in some strains with another sugar (xylose), a sugar alcohol (sorbitol), or both. In contrast, among the dairy probiotic candidates, Lactococcus lactis H61, which has beneficial effects for human skin when drunk as part of fermented milk, was the only strain that showed suppression of acid production in the presence of d-allulose. Strain H61 did not metabolize d-allulose. We did not observe suppression of acid production by strain H61 with the addition of xylose or sorbitol, and xylose and sorbitol were not metabolized by strain H61. The acid production of strain H61 after culture in a constituted medium (tryptone-yeast extract-glucose broth) was also suppressed with the addition of d-allulose, but growth efficiency and sugar fermentation style were not altered. Probiotic activities-such as the angiotensin-converting enzyme inhibitory activity of H61-fermented milk and the superoxide dismutase activity of H61 cells grown in tryptone-yeast extract-glucose broth-were not affected by d-allulose. d-Allulose may suppress acid production in certain lactic acid bacteria without altering their probiotic activity. It may be useful for developing new probiotic dairy products from probiotic strains such as Lactococcus lactis H61.

Effects of ingesting milk fermented by Lactococcus lactis H61 on skin health in young women: a randomized double-blind study.

We conducted a randomized double-blind trial to evaluate the effects of fermented milk produced using only Lactococcus lactis strain H61 as a starter bacterium (H61-fermented milk) on the general health and various skin properties of young women. Healthy female volunteers (n=23; age=19-21r) received H61-fermented milk (10(10) cfu of strain H61/d) or conventional yogurt (10(10) cfu of both Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus per day), as a reference food, daily for 4 wk. Before and at the end of 4 wk, blood samples were taken, and skin hydration (inner forearms and cheek) and melanin content, elasticity, and sebum content (cheek only) were measured. Skin hydration at the inner forearm was higher at wk 4 than at wk 0 in both groups. Sebum content in cheek rose significantly after intervention in the H61-fermented milk group, but not the conventional yogurt group. Other skin parameters did not differ in either group. Serum analysis showed that total protein concentration and platelet count were elevated and reactive oxygen species decreased in both groups after the intervention. Although H61-fermented milk and conventional yogurt had similar effects on skin status and some blood characteristics of participants, an increase of sebum content in cheek is preferable to H61-fermented milk. As skin lipids contribute to maintaining the skin barrier, H61-fermented milk would provide beneficial effects on skin for young women.

Altered superoxide dismutase activity by carbohydrate utilization in a Lactococcus lactis strain.

Reactive oxygen species, such as superoxide, can damage cellular components, such as proteins, lipids, and DNA. Superoxide dismutase (SOD) enzymes catalyze the conversion of superoxide anions to hydrogen peroxide and dioxygen. SOD is present in most lactococcal bacteria, which are commonly used as starters for manufacturing fermented dairy products and may have health benefits when taken orally. We assessed the effects of carbohydrate use on SOD activity in lactococci. In Lactococcus lactis ssp. lactis G50, the SOD activity of cells grown on lactose and galactose was higher than that on glucose; in Lactococcus lactis ssp. cremoris H61, SOD activity was independent of the type of carbohydrate used. We also investigated the activity of NADH oxidase, which is related to the production of superoxide in strains G50 and H61. Activity was highest in G50 cells grown on lactose, lower on galactose, and lowest on glucose, whereas activity in H61 cells did not differ with the carbohydrate source used. The SOD and NADH oxidase activities of strain G50 in three carbohydrates were linked. Strain G50 fermented lactose and galactose to lactate, acetate, formate, and ethanol (mixed-acid fermentation) and fermented glucose to mainly lactate (homolactic fermentation). Strain H61 fermented glucose, lactose, and galactose to mainly lactate (homolactic fermentation). In strain G50, when growth efficiency was reduced by adding a metabolic inhibitor to the growth medium, SOD activity was higher than in the control; however, the metabolism was homofermentative. Aerobic conditions, but not glucose-limited conditions, increased SOD activity, and mixed-acid fermentation occurred. We conclude that the effect of carbohydrate on SOD activity in lactococci is strain dependent and that the activity of commercial lactococci can be enhanced through carbohydrate selection for mixed-acid fermentation or by changing the energy distribution, thus enhancing the value of the starter and the resulting dairy products.

Commensal symbiosis between a Lactococcus lactis strain and an Enterococcus mundtii strain increases cell yield in constituted broth.

To exert their beneficial effects, probiotics need to survive in the stringent conditions of the gastrointestinal tract. Symbiosis between different bacteria is a potential way of enhancing this survival. In developing new probiotic cultures, we investigated the synergic effect between Enterococcus mundtii IFO 13712 and 7 strains of Lactococcus lactis, many of which are widely used as starter bacteria for making dairy products and have probiotic properties. The growth yield of a mixed culture of L. lactis strain Y and IFO 13712 in de Man, Rogosa, and Sharpe broth was greater than that of a single culture. Supernatant from culture of strain IFO 13712 enhanced the growth of strain Y, but that of strain Y did not enhance the growth of strain IFO 13712. This commensalism phenomenon was confirmed by using a simpler tryptone-yeast extract-glucose (TYG) broth. Increased cell yield in mixed culture of the 2 strains compared with single cultures was observed in TYG broth in the presence of both Tween 80 and citrate but not in TYG broth alone or TYG broth containing either Tween 80 or citrate. Thus, the Tween 80 and citrate in the broth contributed to the commensalism. Metabolite analysis revealed that ethanol production in the co-metabolism of glucose and citrate by strain Y was suppressed by mixed culture in TYG broth containing Tween 80 and citrate, compared with that in TYG broth containing citrate alone. The mechanism supporting the observed commensal symbiosis between strains Y and IFO 13712 was the increase in availability of glucose for lactate production by strain Y because, in glycolysis, the pathway from glucose to lactate is energic, whereas the pathway from glucose to ethanol is not. Whether growth stimulation of strain Y by mixing it with IFO 13712 in milk products will enhance the survival of strain Y in the intestine remains to be elucidated.

A derivative of Lactococcus lactis strain H61 with less interleukin-12 induction has a different cell wall.

Lactococcus lactis H61 can increase the cellular immune responses of aged (14-mo-old) senescence-accelerated mice. The aim of this study was to investigate the factors contributing to IL-12 induction by strain H61 by analyzing strains derived from it. Strain H61 derivative no. 13 was obtained by growing the parent strain at 37°C. This derivative induced significantly lower production of IL-12 from J774.1 macrophage cells than did the parent strain H61. The 2 strains differed in the resistance of their whole cells or cell walls to lysozyme, a cell wall-degrading enzyme. Sodium hydroxide treatment to de-O-acetylate muramic acid in the cell walls of the 2 strains reduced the lysozyme resistance, compared with untreated cell walls: at 3h after adding lysozyme, the lysozyme resistance of untreated and NaOH treated cell wall from strain H61 was 55.4% and 11.7%, respectively. The values of untreated and NaOH-treated cell walls from strain no.13 were 73.7 and 42.8%, respectively. The reduction was higher in strain H61, indicating that the cell walls of strain H61 were highly O-acetylated. Trichloroacetic acid treatment to remove wall-associated polymers such as teichoic acids made the lysozyme resistance of the cell walls of both strains similar. The sugar content of cell walls prepared from strain H61 was significantly higher than that of strain no. 13 cell wall. A derivative with less activity for inducing IL-12 by macrophage cells had less O-acetylation and had lower sugar content in the cell wall than did strain H61. Modifying the cell wall of strain H61 may be a useful way to regulate its ability to induce IL-12. Strain H61 has been used as a starter bacterium in the dairy industry. This study could lead to enhancing the value of dairy products made by strain H61 by characterizing the key factor(s) responsible for its stimulation of immunity.

Interaction between Lactococcus lactis and Lactococcus raffinolactis during growth in milk: development of a new starter culture.

Many milk fermentations use mixed cultures of lactic acid bacteria. To select a new mixed starter culture, 100 acid-producing bacterial strains were isolated from raw cow milk. Of these, 13 strains identified as belonging to the genera Lactococcus, Lactobacillus, Leuconostoc, or Weissella (based on phenotypic and genotypic tests) were assessed for a symbiotic effect between pairs of isolated strains during growth in milk. Among the strains tested, a mixed culture of Lactococcus lactis ssp. lactis strain 54 and Lactococcus raffinolactis strain 37 stimulated greater acid production during fermentation than occurred with pure fermentation. This stimulatory effect was not observed in milk supplemented with yeast extract or glucose or in constituted medium. Addition of a cell-free filtrate from milk fermented by strain 54 increased acid production by strain 37; however, the converse effect was not observed. The increased acid production by this mixed culture was, therefore, due to stimulation of strain 37 by metabolic products of strain 54, suggesting that the interaction between strains 54 and 37 is commensal. Analysis with a taste-sensing system indicated that fermented milk containing the mixed culture was more acidic, had more anionic bitterness, had greater aftertastes of anionic bitterness and astringency, and was less salty and umami than milk containing the individual cultures. This study identifies a new commensal relationship between 2 lactococcal strains that are commonly used for making dairy products.

Factors for bile tolerance in Lactococcus lactis: analysis by using plasmid variants.

The factors of bile tolerance (as one among the fundamental characteristics of probiotic bacteria) were determined in lactococci by using plasmid variants. Bile tolerance of Lactococcus lactis wild-type (WT) strains 527 and N7 (determined by viability counts on bile-containing agar) was equivalent to the corresponding plasmid-free derivatives. In contrast, L. lactis WT strain DRC1 had lower bile tolerance than its plasmid-free derivative DRC1021. Plasmid pDR1-1B, extracted from strain DRC1, was introduced into strain DRC1021 by co-transformation with the vector plasmid pGKV21 as an indicator. Strain DRC121 (DRC1021 harboring pGKV21) had good bile tolerance as did strain DRC1021, while strain DRC13 (DRC1021 harboring both pDR1-1B and pGKV21) did not. Fatty acid (FA) composition was different between strains DRC121 and DRC13. The plasmid pDR1-1B or plasmid profile and FA composition are key factors for bile tolerance of strain DRC1, and therefore changing the plasmid profile might be a way of modulating bile tolerance in lactococci.

Different growth media alter the induction of interleukin 12 by a Lactococcus lactis strain.

Lactococcus lactis subsp. lactis G50 has immunomodulatory activity and is a candidate for use as a probiotic strain. We investigated the factors that affect the immunomodulatory activity of this strain. The macrophage-like cell line J774.1A was exposed to live or dead cells of strain G50 grown in different media, and the interleukin (IL) 12 produced by the cell line was then measured. Live cells grown in M17 supplemented with glucose (GM17 cells) induced IL-12 production by J774.1 cells significantly more than did cells grown in deMan Rogosa Sharpe (MRS) broth (MRS cells; P < 0.05). In the case of dead cells, the opposite results were obtained in these two samples. The sugar content of GM17 cells was significantly higher than that of MRS cells (P < 0.01). The fatty acid compositions of GM17 cells and MRS cells differed. Lysis of GM17 cells by lysozyme, which degrades the cell wall, was greater than in MRS cells. The cell wall fraction prepared from GM17 cells induced significantly more IL-12 production than did the fraction from MRS cells (P < 0.05). These results indicated that alterations in cellular components or in the structure of the cell surface by the growth media affected the immunomodulatory activity of strain G50. Attention should be paid to the selection of growth medium in testing for the immunomodulatory activity of lactic acid bacteria.

Phenotypic and molecular characterization of Lactococcus lactis from milk and plants.

AIMS: The aim of this study was to obtain new Lactococcus lactis strains from nondairy materials for use as milk fermentation starters. The genetic and phenotypic traits of the obtained strains were characterized and compared with those of L. lactis strains derived from milk. It was confirmed that the plant-derived bacteria could be used as milk fermentation starters. METHODS AND RESULTS: About 2600 lactic acid bacteria were subjected to screening for L. lactis with species-specific PCR. Specific DNA amplification was observed in 106 isolates. Forty-one strains were selected, including 30 strains of milk-derived and 11 of plant-derived, and their phenotypic traits and genetic profiles were determined. The plant-derived strains showed tolerance for high salt concentration and high pH value, and fermented many more kinds of carbohydrates than the milk-derived strains. There were no remarkable differences in the profiles of enzymes, such as lipases, peptidases and phosphatases. Isolates were investigated by cluster analysis based on randomly amplified polymorphic DNA profiles. There were no significant differences between isolates from milk and those from plant. The L. lactis subsp. cremoris strains were clustered into two distinct groups, one composed of the strains having the typical cremoris phenotype and the other composed of strains having a phenotype similar to subsp. lactis. Fermented milk manufactured using the plant-derived strains were not inferior in flavour to that manufactured using the milk-derived strains. CONCLUSIONS: Plant-derived L. lactis strains are genetically close to milk-derived strains but have various additional capabilities, such as the ability to ferment many additional kinds of carbohydrates and greater stress-tolerance compared with the milk-derived strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The lactic acid bacteria obtained from plants in this study may be applicable for use in the dairy product industry.