Randomized study of Lacticaseibacillus fermented milk in Indonesian elderly houses: Impact on gut microbiota and gut environment

Table 1 Baseline characteristics of the participants, n (%)

	Placebo (n = 54)	Probiotic (n = 56)
Participant type, resident/staff	31 (57.4)/23 (42.6)	34 (60.7)/22 (39.3)
Age, median (IQR), year	65.0 (47.0-61.7)	65.5 (50.8-61.7)
Height, mean ± SD, cm	153.8 ± 10.6	155.0 ± 9.8
Weight, median (IQR), kg	53.9 (41.6-64.6)	52.4 (41.8-66.6)
BMI, median (IQR), kg/m2	22.6 (19.0-25.7)	22.3 (18.6-26.1)
Sex, male/female	15 (27.8)/39 (72.2)	18 (32.1)/38 (67.9)
Nutrient intake, mean ± SD, day
Energy, mean ± SD, kcal	893.30 ± 154.37	901.39 ± 154.51
Water, mean ± SD, g	696.86 ± 134.93	735.78 ± 154.77
Protein, mean ± SD, g	42.28 ± 10.11	41.60 ± 10.31
Fat, mean ± SD, g	38.50 ± 9.03	39.40 ± 10.44
Carbohydrate, mean ± SD, g	95.23 ± 20.55	96.00 ± 15.83
Dietary fiber, mean ± SD, g	5.25 ± 1.93	5.55 ± 1.86
Alcohol, mean ± SD, g	0.00 ± 0.00	0.00 ± 0.01
Polyunsaturated fatty acid, mean ± SD, g	6.87 ± 2.00	7.37 ± 2.56
Cholesterol, mean ± SD, mg	190.50 ± 85.76	202.28 ± 92.92
Vitamin A, mean ± SD, μg	563.72 ± 344.67	536.09 ± 273.61
Carotene, mean ± SD, mg	0.08 ± 0.11	0.09 ± 0.12
Vitamin E, mean ± SD, mg	2.75 ± 0.83	2.81 ± 0.86
Vitamin B1, mean ± SD, mg	0.45 ± 0.15	0.44 ± 0.14
Vitamin B2, mean ± SD, mg	0.52 ± 0.16	0.54 ± 0.15
Vitamin B6, mean ± SD, mg	0.72 ± 0.20	0.74 ± 0.22
Folic acid, mean ± SD, μg	109.34 ± 38.93	110.76 ± 35.88
Vitamin C, mean ± SD, mg	30.02 ± 24.76	30.48 ± 25.23
Sodium, mean ± SD, mg	392.78 ± 375.76	312.71 ± 287.12
Potassium, mean ± SD, mg	1030.62 ± 998.85	919.93 ± 267.01
Calcium, mean ± SD, mg	211.76 ± 96.06	221.41 ± 90.79
Magnesium, mean ± SD, mg	167.40 ± 107.23	159.73 ± 36.73
Phosphorus, mean ± SD, mg	483.79 ± 132.22	469.54 ± 106.26
Iron, mean ± SD, mg	6.02 ± 2.99	5.74 ± 1.50
Zinc, mean ± SD, mg	4.22 ± 0.95	4.35 ± 0.99

IQR: Interquartile range; BMI: Body mass index.

Table 2 Changes in microbiota measured by reverse transcription quantitative polymerase chain reaction

	Placebo						Probiotic
	Baseline		3 months		6 months		Baseline		3 months		6 months
Total bacteria	9.3 ± 1.5	98%	9.9 ± 0.8	98%	9.8 ± 1.4	98%	9.8 ± 0.9	100%	9.9 ± 1.0	98%	9.8 ± 1.0	100%
Bacillota
Clostridium coccoides group	8.8 ± 1.3	89%	9.2 ± 0.9	96%	9.3 ± 0.9	94%	9.0 ± 1.1	100%	9.4 ± 0.7	94%	9.1 ± 1.0	98%
Clostridium leptum subgroup	8.6 ± 1.4	96%	9.2 ± 0.9	94%	9.4 ± 0.7	94%	9.1 ± 1.0	100%	9.1 ± 1.0	98%	9.2 ± 1.0	100%
Clostridium perfringens	6.3 ± 1.5	91%	6.0 ± 1.3	88%	5.5 ± 1.2	92%	6.4 ± 1.4	98%	5.9 ± 1.2	92%	5.5 ± 1.1	94%
Clostridioides difficile	6.0 ± 1.7	6%	6.9 ± 0.9	6%	4.9 ± 1.9	6%	3.9 ± 0.4	5%	< 2.3	0%	< 2.3	0%
Total lactobacilli1	6.4 ± 1.7	93%	7.0 ± 1.3	96%	7.1 ± 1.6	94%	6.7 ± 1.6	98%	7.1 ± 1.3	98%	7.2 ± 1.2	98%
Lactobacillus2	5.4 ± 1.5	76%	5.6 ± 1.4	90%	5.8 ± 1.5	88%	5.7 ± 1.6	88%	5.8 ± 1.5	81%	5.5 ± 1.5	92%
Levilactobacillus brevis2	3.3 ± 0.6	24%	3.5 ± 0.6	37%	3.5 ± 1.2	38%	3.4 ± 0.8	38%	4.0 ± 1.0	23%	3.3 ± 0.4	29%
Lacticaseibacillus2	4.6 ± 1.4	19%	5.2 ± 1.4	31%	4.2 ± 1.0	36%	4.9 ± 1.3	32%	6.1 ± 1.2a	94%b	6.3 ± 0.8b	88%b
Limosilactobacillus fermentum2	5.6 ± 1.0	56%	5.3 ± 0.7	71%	4.9 ± 0.9	56%	5.4 ± 0.7	71%	5.5 ± 0.9	71%	4.9 ± 0.6	65%
Fructilactobacillus fructivorans2	3.1	2%	3.1 ± 0.4	6%	2.7	2%	< 2.3	0%	3.0 ± 0.2	6%	2.8 ± 0.4	6%
Lactiplantibacillus2	4.1 ± 1.1	85%	4.4 ± 1.2	92%	4.6 ± 1.0	94%	4.2 ± 1.0	91%	4.4 ± 1.2	83%	4.6 ± 1.1	98%
Limosilactobacillus except L. fermentum2	4.7 ± 1.3	80%	5.1 ± 1.2	90%	4.7 ± 1.3	94%	5.2 ± 1.3	82%	5.3 ± 1.2	87%	4.9 ± 1.1	90%
Liquorilactobacillus and Ligilactobacillus2	6.8 ± 2.1	35%	7.3 ± 1.6	48%	7.7 ± 1.5	48%	7.1 ± 2.0	36%	6.9 ± 2.0	58%	7.3 ± 1.7	53%
Latilactobacillus2	4.7 ± 0.4	4%	4.6 ± 0.4	15%	4.0 ± 0.8	16%	4.3 ± 0.8	9%	4.0 ± 0.9	12%	3.9 ± 0.8	16%
Enterococcus	6.8 ± 1.5	78%	7.5 ± 0.8	85%	7.5 ± 1.0	86%	6.9 ± 1.1	79%	7.1 ± 1.1c	81%	7.2 ± 1.2	90%
Streptococcus	5.1 ± 1.1	83%	5.0 ± 0.8	96%	5.3 ± 0.8	92%	5.1 ± 0.9	96%	5.0 ± 0.8	94%	5.3 ± 0.9	98%
Staphylococcus	7.4 ± 1.9	70%	8.0 ± 1.0	77%	8.2 ± 0.9	72%	8.1 ± 1.0	80%	8.1 ± 1.2	71%	8.0 ± 1.2	84%
Bacteroidota
Bacteroides fragilis group	7.6 ± 0.9	80%	8.0 ± 0.9	92%	8.2 ± 0.8	94%	7.7 ± 1.0	93%	8.0 ± 0.9	94%	8.2 ± 1.0	98%
Prevotella	8.3 ± 1.7	80%	8.9 ± 1.3	88%	8.9 ± 1.4	74%	8.8 ± 1.2	80%	9.1 ± 1.5	83%	9.1 ± 1.1	78%
Actinomycetota
Atopobium cluster	8.7 ± 1.2	94%	8.9 ± 0.8	96%	8.8 ± 0.9	96%	9.1 ± 0.8	100%	8.9 ± 0.8	98%	8.8 ± 0.8	100%
Bifidobacterium	8.1 ± 1.1	81%	8.0 ± 1.1	94%	8.2 ± 1.1	90%	8.3 ± 1.1	98%	8.5 ± 1.1a	94%	8.1 ± 0.9	98%
Pseudomonadota
Enterobacteriaceae	7.7 ± 1.4	89%	8.2 ± 1.0	96%	8.2 ± 0.9	94%	7.7 ± 1.2	100%	8.1 ± 1.0	96%	8.0 ± 1.0	98%
Pseudomonas	4.3 ± 1.0	30%	3.9 ± 0.9	50%	4.1 ± 0.7	76%	3.8 ± 0.7	27%	3.9 ± 0.9	42%	4.0 ± 0.5	78%

^aP < 0.05 vs placebo.

^bP < 0.01 vs placebo.

^cP < 0.10 vs placebo.

¹Total lactobacilli refers to the sum of bacteria denoted by the bacterial groups.

²The bacterial groups (e.g., Lactobacillus, Levilactobacillus brevis, etc.).

The results are expressed as mean ± SD (log₁₀ cells/g of feces) and detection rate (%).

Table 3 Changes in stool organic acids and amino acid metabolites

	Placebo			Probiotic
	Baseline	3 months	6 months	Baseline	3 months	6 months
Organic acids, μmol/g
Acetate, mean ± SD	13.75 ± 4.82	15.88 ± 8.01	14.63 ± 5.54	14.24 ± 5.49	17.97 ± 8.21	15.81 ± 4.85
Formate, mean ± SD	1.91 ± 0.40	1.71 ± 0.30	2.44 ± 0.24	1.90 ± 0.38	1.72 ± 0.17	2.43 ± 0.21
Propionate, mean ± SD	4.44 ± 2.43	5.56 ± 3.51	5.43 ± 3.81	4.68 ± 2.66	6.46 ± 4.09	5.82 ± 3.27
Isobutyrate, mean ± SD	0.41 ± 0.23	0.50 ± 0.39	0.48 ± 0.28	0.48 ± 0.32	0.51 ± 0.42	0.52 ± 0.27
Butyrate, mean ± SD	3.56 ± 2.23	4.32 ± 3.11	3.95 ± 2.89	4.29 ± 3.03	5.21 ± 3.05b	5.04 ± 3.11a
Isovalerate, mean ± SD	0.36 ± 0.24	0.45 ± 0.35	0.42 ± 0.27	0.44 ± 0.34	0.48 ± 0.39	0.43 ± 0.24
Valerate, mean ± SD	0.65 ± 0.41	0.63 ± 0.47	0.57 ± 0.35	0.73 ± 0.44	0.68 ± 0.39	0.67 ± 0.37
Isocaproate, median (IQR)	0.01 (0.01-0.03)	0.02 (0.01-0.03)	0.01 (0.00-0.01)	0.01 (0.01-0.02)	0.02 (0.01-0.04)	0.01 (0.01-0.01)
Caproate, median (IQR)	0.07 (0.04-0.14)	0.06 (0.01-0.23)	0.05 (0.03-0.12)	0.05 (0.03-0.16)	0.06 (0.02-0.11)	0.05 (0.03-0.17)
Heptanoate, median (IQR)	0.01 (0.01-0.03)	0.01 (0.00-0.10)	0.01 (0.00-0.01)	0.02 (0.01-0.03)	0.00 (0.00-0.06)	0.01 (0.00-0.02)
Lactate, median (IQR)	0.03 (0.02-0.05)	0.03 (0.02-0.04)	0.03 (0.02-0.04)	0.03 (0.01-0.04)	0.02 (0.02-0.03)	0.02 (0.02-0.04)
Succinate, median (IQR)	0.01 (0.01-0.02)	0.02 (0.01-0.04)	0.01 (0.01-0.03)	0.01 (0.01-0.03)	0.03 (0.01-0.06)	0.02 (0.01-0.03)
Total organic acids, mean ± SD	25.31 ± 9.08	29.38 ± 13.83	28.11 ± 11.81	27.00 ± 9.54	33.33 ± 13.86	30.95 ± 10.11
pH
pH, median (IQR)	6.12 (5.86-6.30)	5.99 (5.80-6.35)	6.06 (5.81-6.39)	6.08 (5.75-6.41)	5.89 (5.60-6.38)	5.93 (5.75-6.18)b
Amino acid metabolites, μg/g
Ammonia, median (IQR)	383.7 (245.4-479.5)	467.6 (361.5-568.1)	2015.4 (651.5-3263.7)	358.9 (260.8-506.0)	470.2 (374.6-585.3)	1401.8 (335.8-3116.6)
Indole, median (IQR)	15.92 (9.19-30.40)	13.46 (8.14-22.35)	3.82 (2.78-10.28)	11.97 (7.29-23.70)	14.70 (11.50-23.87)	5.94 (2.95-12.01)
Phenol, median (IQR)	5.24 (4.35-8.67)	5.74 (1.91-14.24)	3.71 (1.96-5.46)	7.02 (2.87-15.46)	3.65 (1.66-10.27)	2.31 (1.40-4.07)
p-cresol, median (IQR)	16.36 (7.42-26.30)	13.03 (8.54-29.98)	6.94 (4.33-11.47)	12.45 (9.46-26.09)	12.44 (7.94-25.91)	8.09 (5.64-12.06)

^aP < 0.05 vs placebo.

^bP < 0.10 vs placebo.

IQR: Interquartile range.

Table 4 Changes in stool frequency, mean ± SD

Months	Stool frequency (times/week)		Stool frequency (days/week)
	Placebo	Probiotic	Placebo	Probiotic
Baseline	6.24 ± 2.61	6.20 ± 2.14	5.59 ± 1.62	5.69 ± 1.63
1	6.05 ± 1.72	6.07 ± 1.43	5.85 ± 1.49	5.89 ± 1.33
2	6.19 ± 1.62	6.27 ± 1.37	5.98 ± 1.38	6.08 ± 1.22
3	6.25 ± 1.47	6.21 ± 1.32	6.02 ± 1.29	6.08 ± 1.26
4	6.18 ± 1.49	6.50 ± 1.14	6.03 ± 1.40	6.27 ± 1.00
5	6.32 ± 1.40	6.56 ± 1.05	6.10 ± 1.27	6.32 ± 0.87
6	6.50 ± 1.25	6.78 ± 1.02	6.29 ± 1.15	6.51 ± 0.77

Table 5 Subgroup analyses on reverse transcription quantitative polymerase chain reaction outcomes

	Participant types	3 months		6 months
		LS mean difference (95%CI)	P value1	LS mean difference (95%CI)	P value1
Total bacteria	Resident	0.12 (-0.33 to 0.57)	0.488	-0.05 (-0.68 to 0.59)	0.752
	Staff	-0.12 (-0.65 to 0.41)		0.11 (-0.62 to 0.83)
Bacillota
Clostridium coccoides group	Resident	0.30 (-0.14 to 0.74)	0.279	0.08 (-0.42 to 0.58)	0.068
	Staff	-0.07 (-0.60 to 0.45)		-0.62 (-1.20 to -0.05)
Clostridium leptum subgroup	Resident	0.13 (-0.35 to 0.61)	0.198	0.06 (-0.42 to 0.54)	0.159
	Staff	-0.36 (-0.93 to 0.22)		-0.47 (-1.03 to 0.09)
Clostridium perfringens	Resident	0.07 (-0.62 to 0.75)	0.451	0.22 (-0.41 to 0.86)	0.491
	Staff	-0.34 (-1.14 to 0.46)		-0.12 (-0.87 to 0.64)
Total lactobacilli	Resident	0.37 (-0.31 to 1.06)	0.273	0.51 (-0.22 to 1.25)	0.067
	Staff	-0.21 (-1.01 to 0.59)		-0.55 (-1.43 to 0.32)
Enterococcus	Resident	-0.13 (-0.70 to 0.43)	0.144	0.32 (-0.26 to 0.90)	0.003b
	Staff	-0.77 (-1.42 to -0.12)		-1.04 (-1.70 to -0.37)
Streptococcus	Resident	-0.06 (-0.47 to 0.36)	0.964	0.08 (-0.37 to 0.53)	0.698
	Staff	-0.07 (-0.56 to 0.42)		-0.06 (-0.57 to 0.46)
Staphylococcus	Resident	0.17 (-0.52 to 0.86)	0.966	-0.01 (-0.65 to 0.62)	0.340
	Staff	0.19 (-0.57 to 0.94)		-0.48 (-1.21 to 0.26)
Bacteroidota
Bacteroides fragilis group	Resident	0.04 (-0.42 to 0.50)	0.639	0.10 (-0.35 to 0.55)	0.167
	Staff	-0.13 (-0.68 to 0.43)		-0.38 (-0.90 to 0.14)
Prevotella	Resident	0.44 (-0.29 to 1.16)	0.206	0.15 (-0.65 to 0.96)	0.911
	Staff	-0.28 (-1.13 to 0.57)		0.22 (-0.61 to 1.04)
Actinomycetota
Atopobium cluster	Resident	0.00 (-0.41 to 0.40)	0.904	-0.05 (-0.49 to 0.38)	0.969
	Staff	-0.04 (-0.53 to 0.44)		-0.07 (-0.57 to 0.44)
Bifidobacterium	Resident	0.84 (0.26 to 1.42)	0.031a	0.25 (-0.26 to 0.75)	0.044a
	Staff	-0.14 (-0.81 to 0.53)		-0.54 (-1.11 to 0.04)
Pseudomonadota
Enterobacteriaceae	Resident	-0.03 (-0.54 to 0.49)	0.390	0.10 (-0.4 to 0.61)	0.102
	Staff	-0.37 (-0.97 to 0.23)		-0.54 (-1.12 to 0.05)
Pseudomonas	Resident	-0.19 (-0.74 to 0.36)	0.721	-0.21 (-0.56 to 0.15)	0.865
	Staff	-0.39 (-1.35 to 0.58)		-0.16 (-0.62 to 0.31)

¹P values for the interaction between treatment and participant type.

^aP < 0.05.

^bP < 0.01.

LS: Least squares; CI: Confidence interval.

Table 6 Number and percentage of participants with adverse events, n (%)

	Placebo	Probiotic	Odds ratio	P value
Any adverse event	22 (40.7)	21 (38.9)	0.87	0.845
Gastrointestinal disorders	8 (14.8)	6 (11.1)	0.69	0.577
Diarrhea	2 (3.7)	2 (3.7)	0.96	1.000
Stomatitis	1 (1.9)	0 (0)	0.32	0.491
Stomachache	2 (3.7)	1 (1.9)	0.47	0.615
Toothache	1 (1.9)	1 (1.9)	0.96	1.000
Pain of epigastrium	1 (1.9)	2 (3.7)	1.96	0.514
Bloated	0 (0)	2 (3.7)	5.00	0.496
Gastric pain	0 (0)	1 (1.9)	2.95	1.000
Dyspepsia syndrome	1 (1.9)	0 (0)	0.32	0.491
Lower abdominal pain	1 (1.9)	0 (0)	0.32	0.491
Nausea	0 (0)	1 (1.9)	2.95	1.000
Nervous system disorders	5 (9.3)	8 (14.8)	1.63	0.557
Musculoskeletal and connective tissue disorders	5 (9.3)	6 (11.1)	1.18	1.000
General disorders and administration site conditions	4 (7.4)	5 (9.3)	1.23	0.523
Respiratory, thoracic, and mediastinal disorders	4 (7.4)	1 (1.9)	0.23	0.202
Infections and infestations	2 (3.7)	2 (3.7)	0.96	1.000
Skin and subcutaneous tissue disorders	2 (3.7)	1 (1.9)	0.47	0.615
Eye disorders	1 (1.9)	1 (1.9)	0.96	1.000
Metabolism and nutrition disorders	0 (0)	1 (1.9)	2.95	1.000
Vascular disorders	0 (0)	1 (1.9)	2.95	1.000
Psychiatric disorders	1 (1.9)	0 (0)	0.32	0.491