Toshiro Sone

Hyaluronic acid production by recombinant Streptococcus thermophilus

Generally recognized as safe, Streptococcus thermophilus was transformed using a plasmid expressing endogenous hyaluronic acid (HA) synthase genes. A single expression of hyaluronic acid synthase (hasA), uridine diphosphate-glucose dehydrogenase gene (hasB), or pyrophosphorylase gene (glmU) and double expression of hasA and hasB were attempted. A streptococcus-Escherichia coli shuttle vector, pBE31, was successfully transfected in S. thermophilus. The single expression of hasA or hasB allowed S. thermophilus to produce about 0.5-1.0 g/l HA. The strains coexpressing of hasA and hasB showed a markedly increased HA production (1.2g/l) which was six-fold increase compared with the wild-type strain. The maximum cell concentration and specific growth rate of each recombinant strain were lower than those of the wild-type strain; however, the specific production rate was more than 100-fold higher. Galactose concentration decreased in the coexpressing strain after depletion of lactose. The bacterial metabolism would be altered in order to achieve a higher production by changing the intracellular metabolism. The average molecular weight of HA (1.0 × 10(6) Da) was not affected by the expression of hasA and hasB. HA produced from recombinant strain could be an alternative material for medical, cosmetic and food utilization instead of HA from conventional pathogenic streptococci.

Streptococcus thermophilus produces exopolysaccharides including hyaluronic acid

Hyaluronic acid (HA) is an important material for medical, cosmetic and food applications. HA is obtained commercially from rooster combs and the fermentation of streptococci. However, the safety problems such as hyaluronidase or exotoxin contamination remain controversial. To reduce the risk of hyaluronidase or exotoxin contamination, we attempted to isolate strains of Streptococcus thermophilus with high productivity of useful exopolysaccharides (EPSs) including HA from traditional dairy food products. Forty-six S. thermophilus strains were isolated from dairy food products, and examined of their HA production using a HA binding protein method. According to the results, six S. thermophilus strains produced EPSs including HA. S. thermophilus YIT 2084 had a markedly high HA productivity (approximately 8 mg/l). We focused on the high-molecular-mass fraction of EPS (2000 kDa) from S. thermophilus YIT 2084. By using high-performance liquid chromatography, it was found that a high-molecular-mass fraction of EPS included N-acetylglucosamine (54.4%) and glucuronic acid (45.6%), which are components of HA. Furthermore, 13C-nuclear magnetic resonance spectroscopy showed that the spectra of the high-molecular-mass fraction corresponded well to those of commercial HA. Here, we described for the first time that S. thermophilus, which is a generally recognized safe bacterium, produces HA. The novel HA-producing bacterium S thermophilus YIT 2084 has great potential for applications in the medical, cosmetic and food fields, although its culture conditions remain to be improved.

Effects of fermentation conditions and soybean peptide supplementation on hyaluronic acid production by Streptococcus thermophilus strain YIT 2084 in milk

To increase the hyaluronic acid (HA) yield from Streptococcus thermophilus YIT 2084, fermentation conditions (pH, temperature, agitation, aeration) were optimized in milk-based medium, and the effects of supplemental soybean peptides, which have different molecular weight distributions, were determined. HA production was enhanced to approximately 100 mg/l at pH 6.8 and 33-40 degrees C. Agitation speed and aeration rate slightly affected HA production. Soybean peptides including those of high molecular weight (approximately 27 to 130 kDa) further increased HA production to 208 mg/l under the optimal condition (pH 6.8, 35 degrees C, 100 rpm), which was 20-fold greater than non-optimal condition. HA production was no longer related to the specific growth rate. The HA produced under the optimal condition included a large amount of high-molecular-weight fraction of 100 to 2000 kDa, compared with under the basal condition without optimization.

Oral administration of Bifidobacterium breve attenuates UV- induced barrier perturbation and oxidative stress in hairless mice skin

Recent studies have shown that some probiotics affect not only the gut but also the skin. However, the effects of probiotics on ultraviolet (UV)-induced skin damage are poorly understood. In this study, we aim to examine whether oral administration of live Bifidobacterium breve strain Yakult (BBY), a typical probiotic, can attenuate skin barrier perturbation caused by UV and reactive oxygen species (ROS) in hairless mice. The mice were orally supplemented with a vehicle only or BBY once a day for nine successive days. Mouse dorsal skin was irradiated with UV from days 6 to 9. The day after the final irradiation, the transepidermal water loss (TEWL), stratum corneum hydration, and oxidation-related factors of the skin were evaluated. We elucidated that BBY prevented the UV-induced increase in TEWL and decrease in stratum corneum hydration. In addition, BBY significantly suppressed the UV-induced increase in hydrogen peroxide levels, oxidation of proteins and lipids, and xanthine oxidase activity in the skin. Conversely, antioxidant capacity did not change regardless of whether BBY was administered or not. In parameters we evaluated, there was a positive correlation between the increase in TEWL and the oxidation levels of proteins and lipids. Our results suggest that oral administration of BBY attenuates UV-induced barrier perturbation and oxidative stress of the skin, and this antioxidative effect is not attributed to enhancement of antioxidant capacity but to the prevention of ROS generation.

Consecutive Intake of Fermented Milk Containing Bifidobacterium breve Strain Yakult and Galacto-oligosaccharides Benefits Skin Condition in Healthy Adult Women

A double-blind, placebo-controlled, randomized trial was conducted to investigate the beneficial effect of probiotic and prebiotic fermented milk on the skin of healthy adult women. Forty healthy Japanese adult female volunteers with healthy skin randomly received either a bottle of probiotic and prebiotic fermented milk containing Bifidobacterium breve strain Yakult and galacto-oligosaccharides (GOS) (active group) or a non-fermented placebo milk containing neither probiotics nor GOS (placebo group) daily for 4 weeks. Before and after intake, hydration levels and cathepsin L-like activity in the stratum corneum and phenol levels in the serum and urine were determined. After intake, the hydration level of the stratum corneum decreased significantly in only the placebo group and was significantly lower than in the active group (p=0.031). Cathepsin L-like activity, an indicator of keratinocyte differentiation, was significantly increased in the active group (p=0.027). Serum and urine phenol levels decreased significantly in the active group (p=0.014, p=0.002, respectively), and serum phenol levels were significantly lower in the active group compared with the placebo group (p=0.006). The consecutive intake of probiotic and prebiotic fermented milk can benefit skin condition without dryness and decrease the levels of phenol production by gut bacteria in healthy adult women.

Photoprotective effects of Bifidobacterium breve supplementation against skin damage induced by ultraviolet irradiation in hairless mice

Probiotics have been considered to affect not only the gut but also the skin. This study aimed at examining whether oral administration of live Bifidobacterium breve strain Yakult (BBY), a typical probiotic, could exert photoprotective effects in hairless mouse skin.

Moisturizing effect of vesicles formed from monoglycerides on human skin

The moisturizing effect of vesicles formed from monoglycerides on human skin was studied by measurement of conductance on and transepidermal waterloss from the skin surface. Although sonication of the monoglycerides in water with Ca2+ gave multilamellar vesicles, the lamellar structure of the vesicles disappeared during their storage without any other additive. With the addition of poly(vinylpyrrolidone) after the sonication, the stability of the vesicles increased and their lamellar structure was maintained for 3 months at 40°C. These vesicles led to a significantly higher water content of the stratum corneum of human skin compared with non‐lamellar monoglyceride, and consequently they improved rough human skin.

Effect of poly(vinylpyrrolidone) on the stability of vesicles formed using monoglyceride

Poly(vinylpyrrolidone) (PVP) stabilized the lamellar structure of vesicles formed using monopalmitin. The electron spin resonance spectrum of spin-labeled PVP suggested that monopalmitin was adsorbed on the vesicles and it formed loops or tails in the lamellar structure. The stabilizing effect of PVP was supposed to be dependent on the steric stabilization of the vesicles and prevention of fusion or aggregation of the vesicles.

Cosmetic Ingredients Fermented by Lactic Acid Bacteria

There is an interesting relationship between the skin and fermentation of lactic acid bacteria (LAB) or bifidobacteria. Supernatants of these bacteria contain lactate and amino acids, which contribute to the hydration of the skin. Many cosmetic ingredients have been developed using LAB and bifidobacteria. In this chapter, four cosmetic ingredients that are being developed are introduced. Skim milk fermented by Streptococcus thermophilus (SE) has skin hydration, antioxidative, and pH control effects. Moreover, the cell protective effect of this ingredient has been proven in recent research. Aloe vera fermented by Lactobacillus plantarum, which was selected from 119 strains of LAB (AE), possesses fourfold greater skin hydration effect than nonfermented A. vera juice. Soybean milk fermented by Bifidobacterium breve has the potential to enhance hyaluronic acid production in three-dimensional culture of human cells. S. thermophilus YIT 2084 was proven able to produce hyaluronic acid. Although hyaluronic acid is a conventional cosmetic ingredient, it has the added value of being safe owing to its production using S. thermophilus, which is generally recognized as safe. It is believed that the technology introduced here will be useful for the development of next-generation cosmetic ingredients .