Heat-killed Lacticaseibacillus paracasei GMNL-653 ameliorates human scalp health by regulating scalp microbiome

Abstract

Background: The equilibrium of the scalp microbiome is important for maintaining healthy scalp conditions, including sebum secretion, dandruff, and hair growth. Many different strategies to improve scalp health have been reported; however, the effect of postbiotics, such as heat-killed probiotics, on scalp health remains unclear. We examined the beneficial effects of heat-killed probiotics consisting of Lacticaseibacillus paracasei, GMNL-653, on scalp health.

Results: Heat-killed GMNL-653 could co-aggregate with scalp commensal fungi, Malassezia furfur, in vitro, and the GMNL-653-derived lipoteichoic acid inhibited the biofilm formation of M. furfur on Hs68 fibroblast cells. The mRNA of hair follicle growth factors, including insulin-like growth factor-1 receptor (IGF-1R), vascular endothelial growth factor, IGF-1, and keratinocyte growth factor was up-regulated in skin-related human cell lines Hs68 and HaCaT after treatment with heat-killed GMNL-653. For clinical observations, we recruited 22 volunteer participants to use the shampoo containing the heat-killed GMNL-653 for 5 months and subsequently measured their scalp conditions, including sebum secretion, dandruff formation, and hair growth. We applied polymerase chain reaction (PCR) to detect the scalp microbiota of M. restricta, M. globosa, Cutibacterium acnes, and Staphylococcus epidermidis. A decrease in dandruff and oil secretion and an increase in hair growth in the human scalp were observed after the use of heat-killed GMNL-653-containing shampoo. The increased abundance of M. globosa and the decreased abundance of M. restricta and C. acnes were also observed. We further found that accumulated L. paracasei abundance was positively correlated with M. globosa abundance and negatively correlated with C. acnes abundance. S. epidermidis and C. acnes abundance was negatively correlated with M. globosa abundance and positively correlated with M. restricta. Meanwhile, M. globosa and M. restricta abundances were negatively associated with each other. C. acnes and S. epidermidis abundances were statistically positively correlated with sebum secretion and dandruff, respectively, in our shampoo clinical trial.

Conclusion: Our study provides a new strategy for human scalp health care using the heat-killed probiotics GMNL-653-containing shampoo. The mechanism may be correlated with the microbiota shift.

Keywords: Heat-killed probiotics; Lacticaseibacillus paracasei; Scalp health care; Scalp microbiome; Shampoo.

Fig. 1

Heat-killed GMNL-653 causes co-aggregation with M. furfur and inhibits its adhesion to Hs86 cells. A, B Heat-killed GMNL-653 (2 × 10⁹ cells/ml), live M. furfur (2 × 10⁹ cfu/ml), or mixtures of heat-killed GMNL-653 and live M. furfur with the ratio of 1:1 were added into tubes and stand at room temperature for 40 min to observe the formation of precipitates (A). Liquids of the suspended area from the tubes of M. furfur alone, GMNL-653 alone, and M. furfur-GMNL mixture were collected after mixing for 0, 20, and 40 min. The liquid was detected by the absorbance at a wavelength of 590 nm (B). The aggregation ability was quantified by the formula described in the Materials and Methods section. C The aggregation of GMNL-653 and M. furfur from (A) was visualized by SEM. D M. furfur were seeded into wells of a 96-well-plate with or without adding 25, 50 µg/ml GMNL-653 derived LTA for 24 h. The biofilm was visualized after staining with 0.1% crystal violet, dissolved with DMSO, and quantified by the absorbance at a wavelength of 590 nm. *, p < 0.05

Fig. 2

Treatment of heat-killed GMNL-653 in Hs68 fibroblasts increases the mRNA expressions of growth factors. 1.5 × 10⁵ cells/well of human fibroblast Hs68 cells were seeded in wells of 6-well-plate for attachment and then cultured with serum free medium for 24 h. Cells were treated with indicated concentration of heat-killed GMNL-653 for 24 h. The mRNA expression of IGF-1R (A), VEGF (B), IGF-1 (C), and KGF (D) were determined by quantified RT-PCR (n = 5). Data were presented as the relative fold changes (mean ± SEM) in compared to non-GMNL-653 treatment control (Ctrl) after normalization with the house-keeping of β-actin. *p < 0.05

Fig. 3

Heat-killed GMNL-653 increases the mRNA expressions of growth factors in HaCaT keratinocytes. Human epidermal keratinocyte HaCaT cells were seeded in 6-well-plate as a cell density of 3 × 10⁵ cells/well and cultured with serum free medium after attachment for 24 h. Cells were treated with the indicated concentrations of heat-killed GMNL-653 for further 24 h. The mRNA expressions of IGF-1R (A), VEGF (B), and IGF-1 (C) were determined by qRT-PCR. Data were presented as the relative fold changes (mean ± SEM) in compared to non-GMNL-653 treatment control (Ctrl) after normalization with the house-keeping of β-actin. *p < 0.05

Fig. 4

The design of a clinical trial with the application of heat-killed GMNL-653-containing shampoo and examinations of scalp conditions among recruited participants. A All participants were requested to use the shampoo without heat-killed GMNL-653 (control shampoo) for the first month. Then, heat-killed GMNL-653-containing shampoo was used for the following 4 month. The collection time points of scalp conditions and scalp microbiota data were indicated by tick. B Data of oil count and hair volume were collected from front, middle, and back of scalp using Sebumeter 815 and Aram TSII. C Data of dandruff were collected from whole scalp by dandruff tapes and analyzed by Image J. Scalp samples for microbiota analysis were collected from whole scalp by wet cotton swabs containing 1 ml PBS buffer with 0.1% Triton X

Fig. 5

The sebum secretion is decreased in human scalp after using heat-killed probiotics GMNL-653-containing shampoo. Oil counts were collected from all participants scalp at the beginning (0 M), after using control shampoo (Ctrl) for 1 month (1 M), and followed using GMNL-653 containing shampoo (GMNL-653) for 1, 2, and 4 months (2 M, 3 M, and 5 M). Oil counts of the front, middle, and back regions of scalp were detected from each participant and calculated the sum values. A Oil counts of all participants (n = 20) were shown. B Oil counts of 7 participants scalp with high oil condition (> 400 µg/cm²) in the beginning were shown. C Oil counts from 13 participants with normal oil condition in the beginning were shown.* p < 0.05 compared with 0 M group. # p < 0.05 compared with the Ctrl group

Fig. 6

Dandruff formation is decreased in human scalp after using heat-killed GMNL-653-containing shampoo. Dandruff from participants were collected using dandruff tapes in the beginning (0M), after using control shampoo for 0.5 and 1 month (0.5M and 1M), and followed using heat-killed GMNL-653 shampoo for 0.5, 1, and 2 months (1.5M, 2M, 3M). A Dandruff level from all participant (n = 18) were shown. B Dandruff level of 8 participants with high dandruff condition (> 0.1%) at the beginning were shown. C Dandruff level of 10 participants with normal condition at the beginning were shown. * p < 0.05 compared with 0 M group. # p < 0.05 compared with the Ctrl group

Fig. 7

The hair volume is increased in human scalp after using heat-killed GMNL-653-containing shampoo. Hair level from participants were collected at the beginning (0M), after using control shampoo for 1 month (1M), and followed using GMNL-653 containing shampoo for 2 and 4 months (3M and 5M). The front, middle, and back regions of scalp in each participant were detected by Aram TSII. Hair volumes were presented as the sum of value of front, middle, and back scalps from each participant. A Hair volumes from all participant (n = 19) were shown. B Hair volumes of the 10 participants with less hair condition (< 125 hairs/cm²) at the beginning were shown. C Hair volumes of the 9 participants with normal hair condition (> 125 hairs/cm²) at the beginning were shown. * p < 0.05 compared with 0 M group. # p < 0.05 compared with the Ctrl group

Fig. 8

The abundance of fungal and bacterial microbiota on human scalp after using heat-killed GMNL-653-containing shampoo. Microbiota samples of each participant were collected from the whole scalp by wet cotton swabs with 0.1% Triton X /PBS buffer at the beginning (Start), after using control shampoo for 1 month (Ctrl), and followed using GMNL-653 shampoo for 1 months (GMNL-653). The liquid samples in cotton swabs were centrifuged to collect the pellets followed by extracting DNA for microbiota analysis. The accumulation of L. paracasei (A) and the abundance of C. acnes (B), S.epidermidis (C), M. globosa (D), and M. restricta (E) in Start, Ctrl, and GMNL-653 groups were quantified by qPCR method. The relative quantified data of L. paracasei, C. acnes, and S. epidermidis were normalized with total bacteria value (A-C) and M. globosa and M. restricta were normalized with total Malassezia value (D-E). * p < 0.05, and ** p < 0.01 compared with Start group. # p < 0.05 compared with the Ctrl group

Fig. 9

The correlations of C. acnes or S. epidermidis to human scalp conditions after using heat-killed GMNL-653-containing shampoo. The levels of C. acnes and S. epidermidis of all participants in three time points after using heat-killed GMNL-653 shampoo (0, 1, 2 months, n = 66) were used for analyzing the Pearson correlation coefficient (R) to oil counts. R and the p value were calculated by SPSS statistics