J Food Bioact

Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online

Journal website www.isnff-jfb.com

Original Research

Volume 9, March 2020, pages 58-69

Low frequency, high power ultrasound: a non-thermal green technique improves phenolic fractions (free, conjugated glycoside, conjugated esters and bound) in fermented seabuckthorn beverage

Figure 1. Phenolic fractional profile (free, bound, glycoside and ester conjugated (a) and free vs bound, glycoside and ester conjugated (b) of defatted whole SB using traditional (TEM), 10 min US (US10), and 15 min US (US15) assisted extractions.

Figure 2. Seabuckthorn puree (P) without and with ultrasound treatment (P+US) on day 0 and 21.

Figure 3. Kombucha (K) samples without and with sugar (S) and without and with ultrasound treatment (US) on day 0, 5 and 21.

Figure 4. Total microbial count (log CFU/mL) of samples with and without US on day 0, 2, 5, 14 and 21.

**Table 1.** Chemical and nutritional analysis of seabuckthorn berries
Fruit	No-US	After US
CAE: Chlorogenic acid equivalent US: Ultrasound.
Moisture%	80.00	78.80
Juice (Yield %)	66.10	65.90
pH	2.60–2.80	2.60–3.20
Acidity%	2.80–2.90	2.70–2.80
°Brix (TSS)	8.20	8.30
TPC (mg CAE/100g)	550	600
Synersis	After 2 hr	After 4 weeks

Table 1. Chemical and nutritional analysis of seabuckthorn berries

Fruit

No-US

After US

CAE: Chlorogenic acid equivalent US: Ultrasound.

Moisture%

80.00

78.80

Juice (Yield %)

66.10

65.90

2.60–2.80

2.60–3.20

Acidity%

2.80–2.90

2.70–2.80

°Brix (TSS)

8.20

8.30

TPC (mg CAE/100g)

550

600

Synersis

After 2 hr

After 4 weeks

**Table 2.** Free, bound, and conjugated phenolic acids and flavonoids in SB berries (mg/kg of dry matter)
	Phenolic acids	Flavonoids	Total
Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05). ND: not determined.
Traditional
Free	ND	20.7 ± 2.3^b	ND	ND	ND	39.0 ± 7.8^a	23.2 ± 5.4^bc	18.9 ± 6.5^d	109.0 ± 12.9^de
Bound	7.7 ± 1.0^d	139.6 ± 5.3^a	5.4 ± 0.8^b	39.3 ± 2.4^a	9.6 ± 1.8^bc	ND	ND	ND	201.5 ± 5.8^cd
Conjugated
Esters	ND	24.8 ± 5.0^b	2.0 ± 0.8^b	7.2 ± 3.2^c	33.9 ± 7.0^a	32.5 ± 5.5^a	ND	ND	100.3 ± 6.7^de
Glycosides	1,492.7 ± 184.5^a	ND	52.5 ± 27.5^a	3.4 ± 0.9^c	ND	ND	26.5 ± 3.7^b	68.6 ± 9.0^b	1,643.7 ± 176.1^a
Total	1,500.4	185.1	59.9	49.8	43.5	71.5	49.7	94.6	2,054.5
US-10
Free	ND	3.7 ± 0.5^e	ND	ND	ND	23.4 ± 3.5^b	47.6 ± 0.1^a	37.7 ± 0.1^c	112.3 ± 4.0^de
Bound	4.5 ± 1.1^d	13.6 ± 2.9^c	1.6 ± 0.2^b	26.7 ± 6.5^b	6.5 ± 0.4^bc	ND	ND	ND	52.9 ± 9.0^e
Conjugated
Esters	ND	6.9 ± 1.2^de	0.7 ± 0.1^b	2.7 ± 0.8^c	11.9 ± 2.7^b	10.0 ± 1.1^c	ND	ND	32.3 ± 1.8^e
Glycosides	212.1 ± 2.9^c	25.3 ± 0.1^b	19.4 ± 5.8^b	ND	ND	ND	ND	ND	256.8 ± 5.0^c
Total	216.5	49.6	21.7	29.5	18.4	33.4	47.6	37.7	454.3
US-15
Free	ND	8.7 ± 0.9^cde	ND	ND	ND	15.2 ± 0.7^c	19.4 ± 1.9^c	15.6 ± 2.0^d	58.9 ± 4.1^e
Bound	6.6 ± 3.0^d	13.6 ± 3.0^c	1.6 ± 0.3^b	28.1 ± 4.1^b	5.4 ± 0.2^c	ND	ND	ND	55.3 ± 9.8^e
Conjugated
Esters	0.6 ± 0.1^d	11.7 ± 2.8^cd	0.7 ± 0.4^b	7.8 ± 0.4^c	31.6 ± 0. 1^a	16.2 ± 0.6^bc	ND	ND	68.5 ± 3.0^e
Glycosides	1,080.1 ± 48.5^b	ND	ND	ND	ND	ND	21.4 ± 2.9^bc	85.4 ± 9.2^a	1,186.9 ± 40.4^b
Total	1,087.3	34.0	2.2	36.0	37.0	31.4	40.8	100.9	1,369.7

Table 2. Free, bound, and conjugated phenolic acids and flavonoids in SB berries (mg/kg of dry matter)

Phenolic acids

Flavonoids

Total

Gallic acid

Proto catechuic acid

Vanillic acid

P-coumaric acid

Ferulic acid

Rutin

Quercetin

Kaempferol

Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05). ND: not determined.

Traditional

Free

20.7 ± 2.3^b

39.0 ± 7.8^a

23.2 ± 5.4^bc

18.9 ± 6.5^d

109.0 ± 12.9^de

Bound

7.7 ± 1.0^d

139.6 ± 5.3^a

5.4 ± 0.8^b

39.3 ± 2.4^a

9.6 ± 1.8^bc

201.5 ± 5.8^cd

Conjugated

Esters

24.8 ± 5.0^b

2.0 ± 0.8^b

7.2 ± 3.2^c

33.9 ± 7.0^a

32.5 ± 5.5^a

100.3 ± 6.7^de

Glycosides

1,492.7 ± 184.5^a

52.5 ± 27.5^a

3.4 ± 0.9^c

26.5 ± 3.7^b

68.6 ± 9.0^b

1,643.7 ± 176.1^a

Total

1,500.4

185.1

59.9

49.8

43.5

71.5

49.7

94.6

2,054.5

US-10

Free

3.7 ± 0.5^e

23.4 ± 3.5^b

47.6 ± 0.1^a

37.7 ± 0.1^c

112.3 ± 4.0^de

Bound

4.5 ± 1.1^d

13.6 ± 2.9^c

1.6 ± 0.2^b

26.7 ± 6.5^b

6.5 ± 0.4^bc

52.9 ± 9.0^e

Conjugated

Esters

6.9 ± 1.2^de

0.7 ± 0.1^b

2.7 ± 0.8^c

11.9 ± 2.7^b

10.0 ± 1.1^c

32.3 ± 1.8^e

Glycosides

212.1 ± 2.9^c

25.3 ± 0.1^b

19.4 ± 5.8^b

256.8 ± 5.0^c

Total

216.5

49.6

21.7

29.5

18.4

33.4

47.6

37.7

454.3

US-15

Free

8.7 ± 0.9^cde

15.2 ± 0.7^c

19.4 ± 1.9^c

15.6 ± 2.0^d

58.9 ± 4.1^e

Bound

6.6 ± 3.0^d

13.6 ± 3.0^c

1.6 ± 0.3^b

28.1 ± 4.1^b

5.4 ± 0.2^c

55.3 ± 9.8^e

Conjugated

Esters

0.6 ± 0.1^d

11.7 ± 2.8^cd

0.7 ± 0.4^b

7.8 ± 0.4^c

31.6 ± 0. 1^a

16.2 ± 0.6^bc

68.5 ± 3.0^e

Glycosides

1,080.1 ± 48.5^b

21.4 ± 2.9^bc

85.4 ± 9.2^a

1,186.9 ± 40.4^b

Total

1,087.3

34.0

2.2

36.0

37.0

31.4

40.8

100.9

1,369.7

**Table 3.** Moisture, ash, protein and fat contents as percentage of P and P+US
Sample	Moisture	Ash	Protein	Fat content pulp (%)	Fat content seed (%)
P: purée with 30% water; US: ultrasonic treatment. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).
P	87.5 ± 0.2^a	1.52 ± 0.00^a	9.80 ± 0.46^a	19.04 ± 0.08^a	14.81 ± 0.08^a
P+US	88.7 ± 1.8^a	1.51 ± 0.00^a	9.84 ± 0.85^a	20.97 ± 0.29^b	15.83 ± 0.28^b

Table 3. Moisture, ash, protein and fat contents as percentage of P and P+US

Sample

Moisture

Ash

Protein

Fat content pulp (%)

Fat content seed (%)

P: purée with 30% water; US: ultrasonic treatment. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).

87.5 ± 0.2^a

1.52 ± 0.00^a

9.80 ± 0.46^a

19.04 ± 0.08^a

14.81 ± 0.08^a

P+US

88.7 ± 1.8^a

1.51 ± 0.00^a

9.84 ± 0.85^a

20.97 ± 0.29^b

15.83 ± 0.28^b

**Table 4.** Rancimat results (Induction time, h) as a measure of oxidative stability for samples with and without US
Sample	Induction time
P: purée with 30% water; US: ultrasonic treatment; K: kombucha; S: sugar. Values are means of triplicates ± standard deviation. The same letters indicate no significant difference using one-way ANOVA (significance: P < 0.05).
P	2.66 ± 0.35^a
K	0.10 ± 0.01^b
K+S	0.13 ± 0.02^b
P + US	2.91 ± 1.46^a
K+US	0.22 ± 0.10^b
K+S+US	0.40 ± 0.28^b

Table 4. Rancimat results (Induction time, h) as a measure of oxidative stability for samples with and without US

Sample

Induction time

P: purée with 30% water; US: ultrasonic treatment; K: kombucha; S: sugar. Values are means of triplicates ± standard deviation. The same letters indicate no significant difference using one-way ANOVA (significance: P < 0.05).

2.66 ± 0.35^a

0.10 ± 0.01^b

K+S

0.13 ± 0.02^b

P + US

2.91 ± 1.46^a

K+US

0.22 ± 0.10^b

K+S+US

0.40 ± 0.28^b

**Table 5.** ORAC (µmol TE/L) results for samples with and without US on day 0,2,5,7 and 21
ORAC	Day
N/A: not applicable; P: purée with 30% water; K: kombucha; S: sugar; US: ultrasonic treatment. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).
P	305.96 ± 0.29^b	N/A	N/A	300.83 ± 3.89^b	298.30 ± 2.35^ab
K	N/A	301.18 ± 3.24^a	299.51 ± 8.07^a	N/A	280.01 ± 7.18^c
K+S	N/A	302.23 ± 1.33^a	286.01 ± 4.94^b	N/A	283.39 ± 14.06^c
P+US	315.42 ± 1.76^a	N/A	N/A	309.99 ± 0.85^a	300.40 ± 1.58^ab
K+US	N/A	311.65 ± 15.12^a	305.87 ± 9.95^a	N/A	308.98 ± 8.15^a
K+S+US	N/A	298.12 ± 4.66^a	282.66 ± 11.22^b	N/A	289.09 ± 5.97^ab

Table 5. ORAC (µmol TE/L) results for samples with and without US on day 0,2,5,7 and 21

ORAC

Day

N/A: not applicable; P: purée with 30% water; K: kombucha; S: sugar; US: ultrasonic treatment. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).

305.96 ± 0.29^b

N/A

300.83 ± 3.89^b

298.30 ± 2.35^ab

N/A

301.18 ± 3.24^a

299.51 ± 8.07^a

N/A

280.01 ± 7.18^c

K+S

N/A

302.23 ± 1.33^a

286.01 ± 4.94^b

N/A

283.39 ± 14.06^c

P+US

315.42 ± 1.76^a

N/A

309.99 ± 0.85^a

300.40 ± 1.58^ab

K+US

N/A

311.65 ± 15.12^a

305.87 ± 9.95^a

N/A

308.98 ± 8.15^a

K+S+US

N/A

298.12 ± 4.66^a

282.66 ± 11.22^b

N/A

289.09 ± 5.97^ab

**Table 6.** Vitamin C concentration (g/L) for samples with and without US
Sample	Vitamin C
P: purée with 30% water; US: ultrasonic treatment; K: kombucha; S: sugar. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).
P	2.31 ± 0.83^a
K	0.20 ± 0.02^b
K+S	0.15 ± 0.00^b
P + US	1.65 ± 0.12^a
K+US	0.10 ± 0.03^b
K+S+US	0.19 ± 0.10^b

Table 6. Vitamin C concentration (g/L) for samples with and without US

Sample

Vitamin C

P: purée with 30% water; US: ultrasonic treatment; K: kombucha; S: sugar. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).

2.31 ± 0.83^a

0.20 ± 0.02^b

K+S

0.15 ± 0.00^b

P + US

1.65 ± 0.12^a

K+US

0.10 ± 0.03^b

K+S+US

0.19 ± 0.10^b

**Table 7.** Water Solubility Index (%) and Water Absorption Index (g/g) of freeze dried samples without and without US
Sample	Water Solubility Index	Water Absorption Index
P: purée with 30% water; K: kombucha; US: ultrasonic treatment; S: sugar. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).
P	46.81 ± 3.03^d	6.64 ± 0.70^b
K	61.60 ± 1.65^c	3.97 ± 0.75^c
K+S	83.31 ± 1.34^a	1.19 ± 0.02^d
P + US	44.62 ± 1.93^d	9.29 ± 1.56^a
K+US	59.25 ± 0.49^c	4.62 ± 2.58^bc
K+S+US	89.93 ± 5.42^a	1.39 ± 0.18^d

Table 7. Water Solubility Index (%) and Water Absorption Index (g/g) of freeze dried samples without and without US

Sample

Water Solubility Index

Water Absorption Index

P: purée with 30% water; K: kombucha; US: ultrasonic treatment; S: sugar. Values are means of triplicates ± standard deviation. The same letters in a column indicate no significant difference using one-way ANOVA (significance: P < 0.05).

46.81 ± 3.03^d

6.64 ± 0.70^b

61.60 ± 1.65^c

3.97 ± 0.75^c

K+S

83.31 ± 1.34^a

1.19 ± 0.02^d

P + US

44.62 ± 1.93^d

9.29 ± 1.56^a

K+US

59.25 ± 0.49^c

4.62 ± 2.58^bc

K+S+US

89.93 ± 5.42^a

1.39 ± 0.18^d