a total of thirty healthy women ( age 3162 years ) from our institute were enrolled in 
 this study . the study was performed between october 2010 and december 2010 as a 
 randomised , double - blind placebo - controlled application test , in two groups , consisting of 
 fifteen women each . exclusion criteria were pregnancy , breast - feeding and those who 
 routinely used medical products for various diseases including metabolic , cardiovascular 
 and hepatic diseases . volunteers were allocated to three age - range categories : 30s ( 351 
 ( se 108 ) years ) , 40s ( 447 ( se 086 ) years ) and 5060s ( 545 
 ( se 161 ) years ) . cremoris h61 , constituted broth ( 05  % meat extract , 05  % yeast 
 extract , 1  % sodium succinate , 1  % sodium chloride and 1  % glucose ) was inoculated to the 
 level of 1  % ( v / v ) with strain h61 ( maff 400007 ) and cultured for 1  d at 35c . bacterial 
 cells were harvested and washed twice with 085  % nacl and then resuspended in the same 
 solution . heat - killed cells were prepared by treatment at 121c for 15  min , followed by 
 centrifugation and lyophilisation . daily doses totalled 20  g and comprised vehicle 
 ( potato starch , lactose and citrate ) with or without 60  mg of lyophilised bacterial cells . the duration of the study was 8 weeks , with assessment of skin parameters at weeks 0 , 4 
 and 8 . this study was conducted according to the guidelines in the declaration of 
 helsinki , and all procedures involving human subjects were approved by the local medical 
 ethics committee at our institute . all test subjects received detailed information listing 
 all parameters relevant to the study , and they submitted a written declaration of consent 
 for participation in the study . hydration of the skin of the right and left inner forearms was measured , and hydration 
 and other parameters of the right and left lateral angles of the cheeks were assessed in 
 the study volunteers . before measurements , each subject washed her cheeks once with soap 
 in tap water and sat quietly for 20  min to equilibrate to the conditions in the testing 
 room ( 22  2 c ; relative humidity , 50  5  % ) . skin hydration was measured by using a corneometer ( cm 825 , courage & 
 khazaka electronics ) 

 . malar skin elasticity was measured by an absorption method ( cutometer 
 sem575 , courage & khazaka electronics ) 

 . in brief , a 2-mm region of skin was aspirated at 300  mbar for 2  s and 
 then released . the 
 melanin and sebum contents in the cheeks were measured by using a mexameter ( courage 
 & khazaka electronics ) 
  and a sebumeter sm810 ( courage & khazaka 
 electronics ) 
 , respectively . skin hydration and melanin content were measured three 
 times at each location and time point . the skin parameters 
 of each subject were measured before intake and , based on those values , the subjects were 
 randomly assigned to either group . the self - evaluation questionnaire comprised two sections to assess perceptions of 
 skin - specific attributes ( section i ) and indictors of general health ( section ii ) at weeks 
 4 and 8 as compared with week 0 , according to their impressions . section i addressed skin 
 properties of lustre , elasticity , darkness , spots , sagging , apparent hair follicles , 
 crevices , wrinkles ( forehead , mouth and eyes ) , dryness , stickiness , freedom from 
 blemishes , redness , ability to retain cosmetics , the number of pimples , size of pimples 
 and the presence of dark circles around eyes . section ii included swelling , 
 oversensitivity to cold , paleness of skin , dizziness , stiff neck , headache , energy level , 
 irritation , constipation , diarrhoea , lustre of hair , appetite , dryness of the throat , 
 sweatiness and ease of falling asleep . subjects evaluated these conditions by responding 
 to questionnaire items by using a five - point scale , in which scores ranged from 2 ( the 
 characteristic was markedly worse than previously ) to 0 ( no change in the characteristic ) 
 to + 2 ( the characteristic was markedly better than previously ) for each item . for all parameters in skin properties and all 
 time points ( weeks 0 , 4 and 8) , descriptive statistics were calculated and were analysed 
 by using a general linear model procedure ( sas version 9.1 ; sas institute ) . least - square 
 means were compared by applying the tukey  kramer test ( pdiff option of the general linear 
 model procedure ) . in addition , the interaction between age , treatment and week was tested . subgroup analyses were performed only when the interaction was significant 
 ( p  <  005 ) or tended towards significance 
 ( 005  <  p  <  01 ) . self - evaluation of skin and body 
 conditions in each subject was analysed by using non - parametric tests ( npar1way procedure 
 of sas ) . the effects of treatment , week and age on all parameters were analysed by using 
 the tukey  kramer test . table 1 indicates the mean measurements 
 associated with various skin parameters in thirty women , as compared according to h61 
 intake , age and week of analysis . age had a significant 
 ( p  =  00162 ) effect only on melanin content in the cheek , which was lower 
 in women in their 30s compared with the other two age groups ( table 1 ) . week ( seasonal change ) had a significant 
 ( p  <  0001 ) effect on all measured parameters except cheek 
 hydration ( table 1 ) . skin hydration of the inner 
 forearm ( p  <  00001 ) , cheek elasticity 
 ( p  =  00014 ) and melanin content ( p  <  00001 ) 
 decreased , whereas sebum content in cheeks ( p  <  00001 ) increased 
 in response to environmental changes , such as temperature and uv exposure , from autumn to 
 winter . table 1.effect of heat - killed cells of lactococcus lactis strain h61 on 
 skin parameters of women ( least - squares means)cheekinner forearm : hydration ( au)hydration ( au)elasticitymelanin ( mi)sebum ( g / cm)treatmentcontrol4055750317167462h614285900323173506 
 p 
 nsnsns00162nsage ( years)30s389596033014147540s41256803251884055060s4475830306181571 
 p 
 nsnsns00162nsweek045157903301881504401587032216854583965810308154757 
 p 
 < 00001ns00014<00001<00001treatment    age : pnsnsnsnsnstreatment    week : pnsnsnsnsnsage    week : pnsns00149nsnstreatment    week    age : p00851nsnsnsnsau , arbitrary units ; mi , melanin index . mean values within a category ( treatment , 
 age and week ) within a column with unlike superscript letters were significantly 
 different ( p  <  005 ) . effect of heat - killed cells of lactococcus lactis strain h61 on 
 skin parameters of women ( least - squares means ) au , arbitrary units ; mi , melanin index .   mean values within a category ( treatment , 
 age and week ) within a column with unlike superscript letters were significantly 
 different ( p  <  005 ) . neither interaction between treatment and age nor interaction between treatment and week 
 had an effect on any parameters of the inner forearm or cheek . a significant 
 ( p  =  00149 ) interaction between age and week was obtained for cheek 
 elasticity , in that women in their 30s had higher elasticity at week 0 than at week 8 
 ( fig . skin elasticity in cheeks was 
 significantly ( p  <  005 ) higher in the youngest group than in the 
 oldest women . 1.skin elasticity of cheeks of women in three age categories ( 30s , -- ; 40s , -- ; 
 5060s , -- ) who received a placebo or heat - killed cells of lactococcus 
 lactis strain h61 . values with unlike letters were significantly different 
 ( p  <  005 ) . skin elasticity of cheeks of women in three age categories ( 30s , -- ; 40s , -- ; 
 5060s , -- ) who received a placebo or heat - killed cells of lactococcus 
 lactis strain h61 . values with unlike letters were significantly different 
 ( p  <  005 ) . interaction between treatment , week and age category on hydration of the inner forearm 
 tended towards significance ( p  =  00851 ; fig . 2 ) . skin hydration in the inner forearm was significantly decreased in all 
 subjects in their 30s in the h61 group and 40s in the placebo and h61 groups at week 4 
 ( p  <  005 ) or week 8 ( p  < 005 ) compared 
 with week 0 . in the subjects in their 30s who received the placebo , skin hydration in the 
 inner forearm at weeks 4 and 8 also decreased compared with week 0 , but these differences 
 were not significant . in contrast , dehydration in 5060-year - old subjects given strain h61 
 cells was suppressed ( p  <  005 ) at weeks 4 and 8 of treatment 
 compared with week 0 . 2.skin hydration of the inner forearm of women in three age categories ( 30s , 40s and 
 5060s ) who received a placebo or heat - killed cells of lactococcus 
 lactis strain h61 . -- , placebo ( 30s ) ;  , h61 ( 30s ) ; -- , placebo 
 ( 40s ) ;  , h61 ( 40s ) ; -- , placebo ( 5060s ) ;  , h61 ( 5060s ) ; au , arbitrary 
 units . .   values with unlike 
 letters were significantly different ( p  <  005 ) . skin hydration of the inner forearm of women in three age categories ( 30s , 40s and 
 5060s ) who received a placebo or heat - killed cells of lactococcus 
 lactis strain h61 . -- , placebo ( 30s ) ;  , h61 ( 30s ) ; -- , placebo 
 ( 40s ) ;  , h61 ( 40s ) ; -- , placebo ( 5060s ) ;  , h61 ( 5060s ) ; au , arbitrary 
 units . values with unlike 
 letters were significantly different ( p  <  005 ) . table 2 indicates the self - scored skin 
 ( elasticity , lustre and apparent hair follicles ) and body ( swelling , dizziness and dryness 
 of the throat ) conditions that yielded significant results concerning the effects of h61 
 treatment . treatment had improvement effects on all of the items in table 2 , although they were not significant . improvement of dizziness was significantly ( p  =  00474 ) affected by age , 
 but a significant difference was not observed in age distribution after analysis using the 
 tukey  kramer test . week had a significant ( p  =  00066 ) effect only on the 
 skin parameter of lustre , which was higher at week 8 than week 4 . table 2.effect of heat - killed cells of lactococcus lactis strain h61 on 
 responses to a self - evaluation questionnaire regarding skin and body condition in 
 women ( least - squares means)skin conditionbody conditionelasticitylustreapparent hair folliclesswellingdizzinessdryness of the throattreatmentcontrol005600280097006700060028h61008002250219011100500160 
 p 
 nsnsnsnsnsnsage ( years)30s01500150010001000100010040s0054012501250042004201135060s000003750250012501250063 
 p 
 nsnsnsns00474nsweek40037011101720067002500528009803220144011100690132 
 p 
 ns00066nsnsnsnstreatment    age : p00984nsns00332nsnstreatment    week : p00086ns0030200125ns00410age    week : pnsnsns00380nsnstreatment    week    age : p0013900753nsnsnsns mean values within a category ( treatment , age and week ) 
 within a column with unlike superscript letters were significantly different 
 ( p  <  005 ) . effect of heat - killed cells of lactococcus lactis strain h61 on 
 responses to a self - evaluation questionnaire regarding skin and body condition in 
 women ( least - squares means )   mean values within a category ( treatment , age and week ) 
 within a column with unlike superscript letters were significantly different 
 ( p  <  005 ) . treatment and age tended to interact in the elasticity of the cheek 
 ( p  =  00984 ) and significantly elasticity of the cheek ( p  =  00086 ) , apparent hair follicles in the 
 cheek ( p  =  00302 ) , swelling ( p  =  00125 ) and dryness of 
 the throat ( p  =  00410 ) all showed significant interaction of treatment 
 and week ( table 2 ) . at week 8 , women who 
 received h61 treatment reported significant ( p  <  005 ) improvement 
 regarding apparent hair follicles in the cheek ( fig . kramer analysis failed to identify significant differences regarding 
 elasticity in the cheek and swelling in subjects . 3.self-evaluation of ( a ) apparent hair follicles and ( b ) dryness of the throat 
 ( score , 2 to + 2 ; relative to week 0 ) in women ( 3162 years ) who received a placebo 
 ( -- ) or heat - killed cells of lactococcus lactis strain h61 
 (  ) . .   mean values with unlike 
 letters were significantly different ( p  <  005 ) . self - evaluation of ( a ) apparent hair follicles and ( b ) dryness of the throat 
 ( score , 2 to + 2 ; relative to week 0 ) in women ( 3162 years ) who received a placebo 
 ( -- ) or heat - killed cells of lactococcus lactis strain h61 
 (  ) . .   mean values with unlike 
 letters were significantly different ( p  <  005 ) . elasticity ( p  =  00139 ; fig . women in the 30s group who 
 received h61 treatment reported a significant ( p  <  005 ) 
 improvement in elasticity at week 8 compared with week 0 and with the placebo group at 
 week 8 . lustre ( p  =  00753 ) also showed a trend toward significance 
 between treatment , week and age , but tukey ( score , 2 to + 2 ; relative to week 0 ) of the cheeks 
 of women in three age categories ( 30s , 40s and 5060s ) who received a placebo or 
 heat - killed cells of lactococcus lactis strain h61 . -- , placebo 
 ( 30s ) ;  , h61 ( 30s ) ; -- , placebo ( 40s ) ;  , h61 ( 40s ) ; -- , placebo 
 ( 5060s ) ;  , h61 ( 5060s ) . values with unlike letters were significantly different 
 ( p  <  005 ) . self - evaluation of elasticity ( score , 2 to + 2 ; relative to week 0 ) of the cheeks 
 of women in three age categories ( 30s , 40s and 5060s ) who received a placebo or 
 heat - killed cells of lactococcus lactis strain h61 . -- , placebo 
 ( 30s ) ;  , h61 ( 30s ) ; -- , placebo ( 40s ) ;  , h61 ( 40s ) ; -- , placebo 
 ( 5060s ) ;  , h61 ( 5060s ) . values with unlike letters were significantly different 
 ( p  <  005 ) . the present study represents an attempt to modulate the skin parameters of women through 
 the intake of heat - killed cells of l. lactis strain h61 and to clarify the 
 effect of this treatment on the skin of women in various age cohorts . oral intake of 40  mg 
 of soya isoflavone aglycones daily has been reported to improve age - associated attributes of 
 skin ( e.g. fine wrinkles and malar skin elasticity ) of middle - aged ( from late 30s to early 
 40s ) japanese women 
 . skin elasticity and the degree of dryness in the cheeks of japanese 
 women ( age 2039 years ) were improved after 4 weeks of consuming yogurt prepared by using 
 lactobacillus delbrueckii subsp . however , no previous reports have addressed the effect of heat - killed 
 lactic acid bacterial cells on human skin properties and age distribution . here , we show 
 that oral intake of heat - killed strain h61 cells was associated with suppression of 
 dehydration due to seasonal change in the inner forearms of women in their 5060s but not in 
 their 30s and 40s . in response to a self - evaluation questionnaire , subjects in their 30s 
 reported significant improvements in elasticity after treatment with h61 cells , compared 
 with that at week 0 , although the results were inconsistent with the data from the 
 mechanical analysis . it is notable that the effect of strain h61 on some skin properties 
 varied according to the age category ( 30s , 40s and 5060s ) . however , the reason for this 
 variation remains unclear as yet . by what mechanism , then , does strain h61 specifically improve the properties of ageing skin 
 in women ? according to responses to the self - evaluation questionnaire , h61 treatment had no 
 effect on general - health items such as constipation , diarrhoea and sleep patterns , which 
 reportedly are associated with skin health 
 . reactive oxygen species , including singlet oxygen and superoxide , are 
 induced in the skin by solar uv radiation and have long been suspected of contributing to 
 the deleterious effects of cutaneous photodamage 
 . reactive oxygen species promote lipid peroxidation , protein oxidation 
 and cross - linking , enzyme inactivation , and dna damage 
 . studies using human dermal fibroblasts have suggested that singlet 
 oxygen mediates uva ( 320400  nm)-induced up - regulation of interstitial collagenase in the 
 skin , through the induction of the inflammatory cytokines il-1 and il-6 
 . oral administration of antioxidant substances such as vitamin 
 c 

  and carotenoids 
  has been shown to have photoprotective effects on the skin . in our 
 preliminary study , heat - killed cells of strain h61 had an antioxidant effect by scavenging 
 the ,-diphenyl--picrylhydrazyl radical . we are currently investigating potential 
 additional antioxidative effects of heat - killed strain h61 , including suppression of 
 oxidative damage of dna and reactive oxygen species generation in macrophage cells . exposure to uvb 
 ( 280320  nm ) can impair specific and non - specific immune responses 
 , and uvb - induced immunomodulation contributes to 
 photocarcinogenesis 
 . in mice , uv exposure reduced the production of t helper type 1 
 cytokines including il-2 and interferon- and increased the synthesis of the t helper type 2 
 cytokines il-4 , il-5 and il-10 
 . in our previous study 
 , spleen cells from mice fed with strain h61 produced more interferon- 
 and il-12 than did those from control mice that did not receive strain h61 , suggesting that 
 administration of strain h61 enhanced t helper type 1 immune responses . therefore , the 
 immunomodulatory activity of strain h61 in enhancing t helper type 1 responses might 
 underlie its ability to improve various properties of ageing skin . further investigation is 
 needed to investigate oxidative status and immune responses by analysing serum from subjects 
 supplemented with the cells of strain h61 , in order to clarify the mechanism of promoting 
 skin health and the differences in the effect of strain h61 among age categories . aged skin has several typical characteristics , including fine wrinkles , sallowness and loss 
 of elasticity 
 . in addition , it has been reported that the secretion of sebum in women 
 reaches a peak during their 20s and declines thereafter 
 . melanin provides varying degrees of brown coloration at the skin 
 surface , and melanin content is affected by internal and external factors such as age , race 
 and sunlight exposure . our present data showed that skin hydration , elasticity and sebum 
 content did not differ among age categories , but melanin content in the cheek varied with 
 age . with regard to skin hydration , although skin in the elderly is generally thought to be 
 dry 

 , adult women ( age 3048 years ) showed higher skin hydration in the cheek 
 and inner forearm than did their children ( age 1014 years ) 
 . in another study 
 , hydration of normal skin did not differ between young and elderly 
 women . the 
 use of instruments that are based on different measurement principles and that assess the 
 skin at different depths and subjects with different ethnic backgrounds may account for the 
 differences reported in the literature . skin disorders such 
 as psoriasis and senile xerosis tend to exacerbate in winter . in japanese women , the 
 hydration state of the stratum corneum on the flexor forearm was significantly lower during 
 winter than during summer 
 . our present study showed similar seasonal changes in skin properties : 
 hydration in the forearm and elasticity and melanin content in the cheek were lower during 
 winter than during autumn , whereas sebum content in the cheek was higher in winter . thermal 
 conditions are apt to affect body temperature regulation and water metabolism in the 
 elderly 
 , and one 's sensitivity to changes in air temperature declines with 
 age 
 . in the present study , changes in environmental factors such as 
 temperature and humidity from autumn to winter affected skin properties in our subjects . furthermore , one study 
  reported that exposed facial skin became more irritated under the 
 influence of the dry and cold environment of winter than did the forearm . however , the 
 present study showed that seasonal changes were apparent in the hydration of the inner 
 forearm but not the cheek . this difference in hydration may be one of the reasons underlying 
 the apparent effect of strain h61 in the inner forearm in this study . repeating the 
 examinations used in the present study but during a different season may help to clarify 
 this issue . in conclusion , we used here a non - invasive mechanical method and self - evaluation survey to 
 show that heat - killed cells of l. lactis strain h61 improved skin 
 properties such as skin hydration and apparent fair follicles in middle - aged and aged 
 japanese women . although the subjects impressions should be given much weight , these 
 impressions regarding the effects of oral intake of strain h61 on skin properties ( e.g. 
 apparent hair follicles ) should be confirmed in future studies by using appropriate 
 measurable instruments .
we conducted a double - blind , placebo - controlled trial to evaluate the effect of 
 heat - killed cells of lactococcus lactis strain h61 on various skin 
 properties of japanese women . 
 volunteers ( age 3162 years ) were randomly assigned to 
 receive test food with or without 60  mg of heat - killed strain h61 ( fifteen women in each 
 group ; h61 and control groups , respectively ) daily for 8 weeks . 
 results were analysed for 
 three age categories ( 30s , 40s and 5060s ) . compared with that at week 0 
 , skin hydration 
 at the inner forearm at weeks 4 and 8 decreased in all volunteers ( except those in their 
 5060s ) because of the environmental change from autumn to winter . 
 the oldest h61 group 
 maintained skin hydration at the inner forearm throughout the study . 
 skin elasticity and 
 melanin content in the cheek decreased and sebum content increased throughout the test 
 period due to seasonal environmental change , regardless of age or h61 treatment . 
 self - evaluation scores for apparent hair follicles and dryness of the throat at week 8 
 were higher in the overall h61 group than in the combined placebo group . the 30s h61 group 
 noted marked improvements in self - surveyed skin elasticity at week 8 compared with at week 
 0 and with the placebo group at week 8 . 
 the results of the present study indicate that 
 oral intake of heat - killed cells of l. lactis strain h61 can improve some 
 skin properties and body characteristics in women . 
 this strain would probably be useful in 
 increasing the quality of life in an ageing population .