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ORIGINAL
Effect of Jersey cow defatted dry milk on salivary volume andcomposition in elderly persons : a pilot study
Tomasz D Pieczonka1,3,‡, Aneta M Bragiel-Pieczonka1,4,‡, Yuko Takeuchi2, Shizuko Yanagisawa2, Masami Yoshioka2,5,
and Yasuko Ishikawa1
Department of Medical Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho,
Tokushima 770-8504, Japan, 2Department of Oral Health Science and Social Welfare, Institute of Biomedical Sciences, Tokushima University
Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan, 3Present adress : Center of New Technologies, University of Warsaw,
Banacha 2c, 02-097 Warsaw, Poland, 4Present adress : Department of Clinical Cytology, Centre of Medical Postgraduate Education, Marymoncka 99/103, 01-813 Warsaw, Poland, 5Present adress : Department of Oral Health Sciences, Faculty of Health and Welfare, Tokushima
Bunri University, 180, Nishihamaboji, Yamashiro-cho, Tokushima 770-8514, Japan, ‡These authors contributed equally to this work
1

Abstract : The administration of Leu57-Leu58-His59-Lys60 (LLHK), Leu58-His59-Lys60 (LHK), and His59-Lys60 (HK) fromβ-lactoglobulin C variant, which is specific to Jersey cow milk, has been shown to prevent and/or restore theage-dependent atrophy and functional decline of salivary glands by affecting gene expression in elderly rats. In
this study, we investigated the effect of Jersey cow defatted milk on salivary volume and composition in elderly
persons. Participants (aged 85 to 98, n = 8) were administered defatted dry milk from Jersey cows twice a day for
4 weeks. Before and after 4 weeks from the start of drinking, saliva was collected and weighed. Salivary cystatin
S and amylase levels were analyzed by Western blotting. To assess the effect of Jersey cow defatted milk on taste
perception, questionnaires were used. Salivary volume after oral administration of 40 g of Jersey cow defatted
dry milk daily for 4 weeks was 1.8 times higher than that before administration. Salivary cystatin S and amylase
levels significantly increased after administration of Jersey cow defatted dry milk. Moreover, all participants
who had taste impairment reported improved taste perception after administration. The administration of Jersey cow defatted dry milk increased salivary volume and changed the composition of saliva in elderly persons.
Furthermore, it improved taste perception. J. Med. Invest. 68 : 280-285, August, 2021
Keywords : elderly person, salivary volume, salivary composition, Jersey cow defatted dry milk, taste perception

INTRODUCTION
Saliva plays many vital roles in oral and systemic health.
Saliva has multiple functions, including cleaning of the oralcavity, dissolution of tastants, dilution and digestion of food,lubrication, moistening of teeth and oropharyngeal mucosa, andfacilitating chewing, bolus formation, swallowing, and speech (1).
Low salivary flow induces not only caries dentium and periodontal disease, but also causes difficulties in speaking, chewing,tasting, and swallowing (2, 3). Periodontal disease increases
the risk of cardiovascular and atherosclerosis (4). Difficulties inchewing and swallowing lead to aspiration pneumonia (5) and
impairment of nutritional status (6). Overall, low salivary flow
decreases the quality of life.
Saliva is secreted from three major salivary glands (parotid, submandibular and sublingual glands) and many minor
salivary glands which are innervated by autonomic nerves (7).
The submandibular and sublingual glands are innervated byparasympathetic nerve derived from the superior salivatory nucleus, while parotid glands are regulated by the nerve supplied
from the inferior salivatory nucleus. Minor salivary glands are
innervated by the parasympathetic fibers of the buccal branch of
the mandibular nerve. Therefore, cholinergic parasympathetic
nerve innervates all of these salivary glands. On the other hand,
Received for publication December 10, 2020 ; accepted May 12, 2021.
Address correspondence and reprint requests to Yasuko Ishikawa,
Department of Medical Pharmacology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15, Kuramoto-cho,
Tokushima 770-8504, Japan. E-mail : ysikawa@mvj.biglobe.ne.jp

adrenergic sympathetic innervation of submandibular and parotid glands is derived from the spinal cord. The sublingual andminor salivary glands supply a sparse adrenergic nerve fiber. As
a result, the stimulation of cholinergic nerve induces a watery,
protein-rich fluid secretion from salivary glands, whereas adrenergic stimulation leads to evoke protein-rich fluid secretion.
In parasympathetic and / or sympathetic nerves, non-adrenergicand non-cholinergic neurotransmitters including substance P,
vasoactive intestinal peptide, neuropeptide Y, neurokinin A, andneuronal nitric oxide synthase regulate nerve activity.
The unstimulated whole saliva and the unstimulated andstimulated saliva from submandibular and sublingual glands
decrease during aging (8, 9). The unstimulated and stimulated
saliva from parotid glands however does not show an age-related
decrease. During aging, the parenchyma of salivary glands isgradually replaced by fat, connective tissue, and oncocytes, resulting in acinar cell atrophy in humans (10) and animals (11).
We previously reported that the administration of whey from
Jersey cow milk mitigates the age-dependent atrophy and functional decline of rat salivary glands, accompanied with changes in gene expression (12). β-Lactoglobulin A and B are themost common variants, while the β-lactoglobulin C variant isobserved in Jersey cattle. There is one amino acid difference
between the B variant (Gln59) and C variant (His59) (13, 14). In
previous experiments, we investigated the effects of Leu57-Leu58His59 -Lys60 (LLHK), Leu58 -His59 -Lys60 (LHK), and His59 -Lys60(HK) peptides on the gene expression profile, age-dependent atrophy, and dysfunction of rat salivary glands. LLHK, LHK, andHK administration prevents and / or restores the age-dependent
atrophy and functional decline of salivary glands by affecting
gene expression (15). In this study, we investigated the effect

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The Journal of Medical Investigation Vol. 68 August 2021
of defatted dry milk from Jersey cows on salivary volume andcomposition in elderly persons.

METHODS
ParticipantsParticipants (aged 85 to 98, 7 women, 1 man) (Table 1) dwelled
in a care house Haiji (Tokushima, Japan). They participated inthis study as volunteers.

F1

F2

Woman

Woman

86

98

Table 2. Compositional characteristics of the defatted dry milk from
Jersey cows

Table 1. Characteristics of participantsParticipant Gender Age

were administered 20 g of defatted dry milk from Jersey cows
(Hiruzen Dairy Farming Farm Co-op, Okayama, Japan) twice
a day for 4 weeks. Compositional characteristics of the defatted
dry milk from Jersey cows are in Table 2. Each time, participants dissolved 20 g of defatted dry milk in 100 mL of hot waterand drank 100 mL. After 4 weeks from the start of drinking,saliva was collected at 2 p.m. as described above and stored at-80°C until further processing.

ConstituentsTreatment/100g

Measurement method

Protein

39.2g

Kjeldahl method

Amlodipine (Ca 2+ Channel Blocker),
Reminyl (Dementia Improving Drug), Neodopaston (Parkinsonism Treatment)

Fat

0.3g

Roese-Gottlieb method

Carbohydrate

49.6g

Deduction method

Merislon (Anti-vertigo Medication), Plavix (Anti-platelet Medication), Mucosta(Mucosal Protection Drug), Berizyme(Digestive Enzyme Preparation), Lasix(Anti-hypertensive Drug), Predonine (Glucocorticoid Medication), Nexium (Proton
Pump Inhibitor), Mucodyne (Mucolytic
Medication)

Ash

7.9g

Direct ashing method

F3

Woman

92

Domperidone (Gastric Prokinetic Agent),
Calfina (Active Vitamin D3), Mevalotin(Lipid-lowering Treatment), Nexium (Proton Pump Inhibitor), Viviant (Estrogen
Receptor Modulator)

F4

Woman

90

Depas (Tranquilizer), Ecabet (Anti-ulcer
Agent), Ferromia (Anmtianemic Agent),
Celecox (Anti-inflammatory Drug)

F5

Woman

85

Ferromia (Anmtianemic Agent)

F6

Woman

81

Rebamipide (Anti-ulcer Agent), Glufast(Rapid-acting Insulin Secretion-stimulating Agent), Basen (Alpha-glucosidase
Inhibitor), Aricept (Acetylcholinesterase
Inhibitor), Warfarin (Antic-oagulant),
Bayaspirin (Anti-inflammatory Drug),
Lasix (Anti-hypertensive Drug), Lanirapid (Cardiac Glycoside), Feburic (Hyperuricemia Treatment), Famotidine (Histamine H2 -receptor Antagonist)

F7

Woman

86

Nicergoline (Senile Dementia Treatment),
Ecabet (Anti-ulcer Agent), Cholebine (Hypercholesterolaemia Treatment), Lipidil(Dyslipidemia Treatment)

M1

Man

87

Casodex (Anti-androgen Medication)

Ethical considerations
The present study was approved by the ethics committee ofTokushima University Hospital (Approval number 2116 in 2014).
Before implementation, all participants were given written andverbal information as to the purpose and method of the study,
protection of personal information, and freedom of consent andwithdrawal. All participants signed the informed consent forms.

Experimental design
Before the experiment, five samples of saliva from participants were collected at 2 p.m. using Salivette (Sarsted Inc.,
Numbrecht, Germany). They gently chewed a Salivette swab for
1 min and the samples were centrifuged at 1,000 g for 10 min (16,17). The weights of supernatants were measured and counted
as secreted saliva (g / min). The saliva was stored in the laboratory at -80°C until further processing. Then, the participants

Calcium

1400mgAtomic absorption
spectrophotometry

Sodium

420mg

Atomic absorption
spectrophotometry

Water

3g

Energy358kcalNormal pressure heat
drying method
Conversion method

Data were analyzed by Public Interest Incorporated Foundation
Okayamakenkenkouzukurizaidan.

Gel electrophoresis and Western blot analysis
Saliva samples were dissolved with Laemmli buffer, and 15µL was subjected to SDS-polyacrylamide gel electrophoresis(PAGE) on 15% and 10% gels. Fifteen % and 10% gels wereused for analyses of cystatin S and amylase, respectively. For
immunoblot analysis, separated proteins were transferred
onto a nitrocellulose membrane (Hybond ECL ; Amersham
Biosciences, Little Chalfont, UK) (16, 17). The membrane wasblocked overnight with 0.1% Tween 20 in Tris-buffered saline(pH 7.4). The blot was then incubated with rabbit anti-cystatin
S antibody (1:500, Sino Biological Inc., Beijing, China) or rabbit
anti-amylase antibody (1:1500, Calbiochem, Darmstadt, Germany) (11). The membrane was washed with Tris-buffered saline
containing 0.1% Tween 20 and incubated with horseradish
peroxidase-linked donkey anti-rabbit IgG, whole Ab (1:5000, GE
Healthcare, Piscataway, NJ, USA) for 1 h. Immunoblots were
processed using enhanced chemiluminescence (ECL) (Amersham), and the signals were visualized on a Chemi Doc apparatus(Bio-Rad, Hercules, CA, USA). The bands were quantified using
Quantity One software (Bio-Rad).

Questionnaires
The following three questions were asked of participants. Q1.
Do you feel that your regular diet tastes good? Q2. Do you chokewhile drinking? Q3. Does your mouth feel dry?

Statistical analysis
Data are expressed as the mean value ± standard error (SE).
To test for statistically significant differences between two
groups, a paired Student’s t test was used for biochemical tests
and Western blot analysis. The statistical significance of relative quantitative changes between two groups was evaluatedby Wilcoxon signed-rank test. p-Values of less than 0.05 wereconsidered statistically significant. Correlation coefficient (r) was
obtained using simple regression analysis (Excel software).

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RESULTS

Effect of Jersey cow defatted dry milk on salivary volume
In all participants, salivary volume after oral administrationof 40 g of Jersey cow defatted dry milk daily for 4 weeks washigher compared to the volume before administration (Fig. 1).
It significantly increased in all participants except one (7 / 8).
The mean ratio (salivary volume after administration / salivary
volume before administration) was 1.8. Participants who had
smaller initial salivary secretions had larger ratios (Fig. 2). The
correlation coefficient between the ratio and the salivary volume
before administration was -0.8604.

Effect of Jersey cow defatted dry milk on salivary composition
To investigate the effect of Jersey cow defatted milk on thelevels of salivary cystatin S, saliva was subjected to Western blot
analysis using anti-cystatin S antibody (Fig. 3, a). In all participants, the cystatin S levels in 15 µL of saliva were increased
after the administration. The bands were quantified using
Quantity One software, and the band density in the 1-min saliva
of each participant was calculated. The density per the 1-min saliva significantly increased after the administration (p = 0.0066)(Fig. 3, b). Since individual differences in the density both before
and after the administration were large, relative quantification
was calculated and shown in Fig. 3, c (p = 0.0117). Jersey cow
defatted milk increased the salivary cystatin S levels by 4.3-foldcompared to before the administration.
To investigate the effect of Jersey cow defatted milk on thelevel of salivary amylase, saliva was subjected to Western blot
analysis using anti-amylase antibody (Fig. 4, a). Amylase levels
in 15 µL of saliva were increased in some participants (F1, F3,
F4, F5, and F7) but decreased in others (F2, F6, and M1) after
the administration. The bands were quantified using Quantity
One software, and the band density in 1-min saliva of eachparticipant was calculated. The density per the 1-min saliva
significantly increased after the administration (p = 0.0331)(Fig. 4, b). Relative quantification was calculated and shown in
Fig. 4, c (p = 0.0357). The amylase levels in whole saliva after theadministration was increased by 1.9-fold compared to that before
the administration.

Figure 1. Salivary volume in participants before and afteradministration of Jersey cow defatted dry milk. Saliva was collected
for 1 min from participants before and after administration of Jersey
cow defatted dry milk daily for 4 weeks, and was centrifuged at1000 g for 10 min. The resultant supernatant was used for volume
measurement. Data represent means ± SE (n = 5). *p < 0.05, **p < 0.01
vs. salivary volume before administration.

Figure 3. Salivary cystatin S levels in participants before and after

Figure 2. Correlation between the ratio and the salivary volume
before administration. The vertical axis shows the ratio (the salivary
volume after administration was divided by the salivary volume
before administration). The horizontal axis shows the salivary volume
before administration.

administration. Saliva was dissolved with Laemmli buffer and 15µL was subjected to SDS-PAGE. The protein was transferred to anitrocellulose membrane and immune blotted with anti-cystatin S
antibody. (a) A typical Western blot was shown in photography. (b)The bands on the blots were analyzed by Quantity One Software.
The band density in 1-min saliva was calculated and expressed in
the graph. Data represent means ± SE (n = 8). **p < 0.01 ; Student’s
t test. (c) Values were expressed as relative quantitative changes.
The amount before administration was assigned to 100%. †p < 0.05 ;Wilcoxon signed-rank test.

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The Journal of Medical Investigation Vol. 68 August 2021
Effect of Jersey cow defatted dry milk on taste perception

cow defatted milk did not influence choking when drinking or
the feeling of dry mouth.

To investigate the effect of Jersey cow defatted milk on taste
perception, questionnaires were used (Table 3). After the administration of the milk, all participants who had previous taste impairment reported improved taste perception, saying that theirmeals were good or pretty good. But the administration of Jersey

Effect of Jersey cow defatted dry milk on side effects
In oral cavity and systemic health, there were not the outstanding side effects caused by the administration of the milk.

DISCUSSION

Figure 4. Salivary amylase levels in participants before and afteradministration. Saliva was dissolved with Laemmli buffer and 15µL was subjected to SDS-PAGE. The protein was transferred to anitrocellulose membrane and immune blotted with anti-amylase
antibody. (a) A typical Western blot is shown in photography. (b) The
bands on the blots were analyzed by Quantity One Software. Theband density in 1-min saliva was calculated and expressed in thegraph. Data represent means ± SE (n = 8). *p < 0.05 ; Student’s t test. (c)Values were expressed as relative quantitative changes. The amount
before administration was assigned to 100%. †p < 0.05 ; Wilcoxon
signed-rank test.

Previously, we reported that the administration of whey from
Jersey cow milk mitigates age-dependent atrophy and functional
decline of salivary glands accompanied with changes in gene
expression in rats (12). We found that the bioactive peptides,
which were LLHK, LHK, and HK derived from β-lactoglobulin
C variant, prevent and / or restore the age-depending decline instructure and function of salivary gland (15). In those experiments, older rats drank the whey or the peptides. In the present
study, elderly people drank commercially available Jersey cow
dry milk. Participants drank 40 g of Jersey cow defatted drymilk daily, which contains about 80 mg of β-lactoglobulin C (14).
Eighty mg of β-lactoglobulin C is converted to 2.5 mg of LLHK,
1.9 mg of LHK, or 1.3 mg of HK. Even though participants we
exposed to small amounts of peptides, these peptides stimulated
salivary secretion and improved the perceived taste of participants’ diets.
The use of multiple medicines is very common in the elderly
persons (18). Many medications induce salivary gland dysfunction, xerostomia, and sialorrhea (19). In all elderly participants
who took multiple pharmaceuticals in this experiment, Jersey
cow dry milk induced higher salivary secretion and made themfeel that their diets tasted better. Jersey cow dry milk induced
salivary secretion even in Participant F1, who used a Ca 2+channel blocker that blocks signal transduction of the M1 andM3 muscarinic receptors or α1-adrenoceptor. The milk induced
salivary secretion also in Participant F6, who used Aricept (anacetylcholinesterase inhibitor) and Famotidine (a histamine
H2-receptor antagonist). As LLHK, LHK, and HK may activate
non-adrenergic and non-cholinergic nerves (15), it seems that
medications had little influence on LLHK, LHK, and HK-induced gene expression.
In general, low-fat milk products have many health benefits,
including the prevention of age-related cognitive decline anddementia (20), a decrease in blood pressure (21), a decreased
risk of diabetes (22), and a decreased risk of stroke and heartdisease (23). Furthermore, these products possess antimicrobial,

Table 3. Participants’ perception before and after the administration of Jersey cow defatted dry milk
Grade

F1
Be → AfF2
Be → Af

F4
Be → Af

F5
Be → Af

F6
Be → Af

F7
Be → Af

M1
Be → Af

4→4

2→3

1→4

4→4

4→4

1→4

2→3

2→3

4→4

4→3

4→4

4→4

4→3

4→4

1→1

1→1

2→2

2→2

2→2

1→1

1→1

1 Bad
Q1 : Do you feelthat your regular
diet tastes good?

2 Somewhat bad
3 Pretty good
4 Good1 OftenQ2 : Do you chokewhile drinking?

2 Sometimes
3 Rarely
4 Never

Q3 : Does yourmouth feel dry?

1 Yes
2 No

Be : before administration, Af : after administration

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TD. Pieczonka, et al. Cure of dry mouth by Jersey cow milk

anticancer, immunomodulatory, and opioid activities (24-26).
Milk proteins consist of many peptides and amino acids that areoften inactive within the sequence of the parent protein, but are
released during food processing and digestion, changing into
biologically active compounds. Branched-chain amino acids (22,27) and peptides such as LKPTPEGDL and LKPTPEGDLEIL
derived from β-lactoglobulin (24) inhibit dipeptidyl peptidase IV
activity, leading to an insulinotrophic effect. ALPMHIR and
WLAHK peptides derived from β-lactoglobulin and α-lactoalbumin respectively inhibit angiotensin-I-converting enzyme
activity, leading to decreased hypertension (25, 26). IIAQK,
GLDKQK, ALPMH, and TPEGDLEILLQK peptides derived
from β-lactoglobulin inhibit cholesterol absorption, resulting indecreasing serum cholesterol levels (25, 28). Peptides such as
YLLF and YGLF, derived from β-lactoglobulin and α-lactoalbumin respectively, behave as µ-opioid receptor agonists, inducing
ileum-contraction and endothelium-independent relaxation (25,
28, 29). The peptide CRRWQWRMKKLGAPSICV, derived fromlactoferrin, possesses antimicrobial activity against oral pathogens (25, 30). Glycomacropeptide, which is derived from κ-casein,
prevents the adhesion of cariogenic bacteria to tooth surfaces,
inhibiting dental plaques and caries (25, 31).
As previously mentioned, oral administration of LHK upregulates proline-rich protein genes (Prpmp5, Prb3, Prp2, Prb1, Prp15),
cystatin genes (Cst5, Cyss, Vegp2), amylase genes (Amy1a, Amy2a3),
and lysozyme gene (Lyzl1), and AP-2 transcription factor gene(Tcfap2b) (15). The administration of LLHK, LHK, and HTK prevents and / or restores the age-dependent atrophy and functional
decline of salivary glands. These peptides, which derived fromβ-lactoglobulin C variant, increase salivary volume and change
the salivary compositions in rats, suggesting that these peptides
are effective in improvement at the quality of human life. This
study showed that the milk including β-lactoglobulin C variant
increased salivary volume, changed the composition of saliva,
and improved taste perception in elderly persons.

CONCLUSIONS
The administration of Jersey cow defatted dry milk increased
salivary volume, changed the composition of saliva, and improved taste perception in elderly persons.

DECLARATION OF CONFLICTING INTERESTS
The authors declare that they have no competing interests.

ACKNOWLEDGEMENTS
We are grateful to the executive director and staff of the carehouse Haiji (Tokushima, Japan) for their acceptance of our protocol. We would like to thank Mrs. Satsuki Yabuuchi for her help
in liaisons and communications between the care house and us.
We also thank Hiruzen Dairy Farming Farm Co-op (Okayama,
Japan) for offering Jersey cow defatted dry milk.

FUNDING
The study was supported by a pilot project program from
Tokushima University Graduate School.

ETHICAL CONSIDERATION AND CONSENT TO
PARTICIPATEAll protocols of this study were approved by the Medical Ethics
Committee of Tokushima University Hospital (Approval number
2116, date of approval : October 27th, 2014) and was conducted inaccordance with the Declaration of Helsinki ethical principles.
Written informed consent from all participants were obtained
prior to start of the study.

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