chapter iv –enrichment and stabilization of...

Chapter IV –Enrichment and stabilization of PUFA

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CHAPTER IV – Enrichment and stabilization of polyunsaturated fatty

acid in recovered lipids from fish visceral waste

n-3 Long-chain polyunsaturated fatty acids (PUFA), especially eicosapentaenoic

acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6), have reported to augments

various biochemical changes in degenerative disorders (Russo, 2009; Mantzioris et al.

2000). There have been many reports regarding improving the concentration of PUFAs in

fish oil and marine algae using methods, such as urea inclusion complexation, molecular

distillation, low temperature fractional crystallization, liquid-liquid extraction-fraction,

high performance liquid chromatographic seperation, and salt solubility methods

(Chakraborty and Paulraj, 2007; Guil-Guerrero et al. 2007). However, most of these

methods are not selective for fatty acids and consume a large amount of energy

(Chakraborty et al. 2010). Concentration of n-3 PUFA by enzymatic processes is based

on the use of specific hydrolytic enzymes, like lipases, which catalyze hydrolysis,

ethanolysis or transesterification reaction of triglycerides. Lipase catalyzed concentration

of n-3 PUFA are promising alternative have shown energy saving and are environmental

friendly technology (Shahidi and Wanasundara, 1998). Due to the fatty acid distribution

in the glycerol backbone of triglycerides and the stereospecific activity of certain lipases

(Ando et al. 1996; Gupta et al. 2004), enzymatic hydrolysis is very useful method both in

n-3 PUFA concentration in fish oil and in the production of structured lipids. In this

study, by lipase hydrolysis was attempted to develop a process for simultaneous recovery

and concentration of PUFA from FVW to obtain PUFA enriched fish oil.

One of the major drawbacks in fish oil is their high susceptibility to oxidation,

which results in formation of toxic products (peroxides or volatile compounds) those are

responsible for the non-desirable off-flavours. This is mainly due to their naturally high

content of EPA and DHA. Nevertheless, they are susceptible to oxidation, which is

associated with their rancidity and loss in nutritive value (Frankel et al. 1998). Apart from

high levels of PUFAs, the presence of heme pigments and trace amounts of metallic ions

makes the fish and fish oil prone to lipid oxidation (Hsieh and Kinsella, 1989). To retard

such a quality loss, synthetic antioxidants have been used to decrease lipid oxidation

during the processing and storage of fish and fish products (Boyd et al. 1993).


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Auto-oxidation of PUFA results in free radical generation through reaction

catalyzed by heat, light, trace metals or enzymes (Wang et al. 2011). The free radicals

formed propagate auto-oxidation by reacting with oxygen which results in formation of

hydroperoxides that breakdown to generate other new free radicals (Wang et al. 2011).

Oxidation of lipids can be prevented or minimized during the processing of FVW by like

enzymatic hydrolysis and fermentation. The commonly used antioxidants in food or food

processing are tocopherol, butylated hydroxytoluene (BHT), butylated hydroxyanisole

(BHA) and tertiary butyl hydroquinone (TBHQ). Butylated hydroxyanisole (BHA) is a

phenolic antioxidant that is generally recognized as safe by the FDA but its use is limited

in foods (CFR, 2007; part 172.110). In animal food processing studies, BHA has been

shown to be carcinogenic at high levels (1250 ppm) (Williams e al. 1999). BHT is a

phenolic antioxidant that is generally recognized as safe by the FDA (CFR, 2007; part

172.110). BHT is a fat soluble antioxidant that acts as a synthetic analogue of vitamin E

(Shahidi and Wanasundara, 1992).

The focus of this chapter was to recover lipids with n-3 PUFA-enriched

acylglycerols during fermentation and enzymatic hydrolysis with lipase aided hydrolysis.

Several microbial lipases were used to screen suitable enzyme for n-3 PUFA enrichment

during the fermentation and enzymatic hydrolysis process. Secondly, the effect of various

antioxidants (α-tocopherol, BHT, BHT and TBHQ) in stabilization of PUFA during the

recovery process was also studied. This chapter deals mainly two aspects :

(i) Enrichment of PUFA by lipases during fermentation and enzymatic hydrolysis of

FVW and

(ii) Stabilization of PUFA during fermentation and enzymatic hydrolysis with

antioxidants.

Results

Enrichment of PUFA by lipase hydrolysis during recovery of lipids by lactic acid

fermentation (LAF) and enzymatic hydrolysis (EH)

Enrichment of PUFA in triglyceride (lipids) was carried out by hydrolyzing SFA

and MUFA in glycerol moiety by sn-1,3-specific lipase (Aspergillus niger, Thermomyces

lanuginose, Mucor javanicus) and non specific lipases (Candida rugosa, Candida

cylindrical) (Table 4.1). Abbreviations of these lipases given in the Table 4.1 are used


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hereafter in the text. These lipases were introduced after 24 h of lactic acid fermentation

by P acidilactici NCIM5368 and 60 min after enzymatic hydrolysis using fungal

protease. The protein hydrolysate formed after 24h of lactic acid fermentation or 60 min

of proteolytic hydrolysis acts as an emulsifier for lipase reaction. The recovered oil was

analyzed for triglyceride fatty acid composition and acid value.

Enzyme activity of the lipases

The activity of lipases used for the concentration of PUFA in fish oil recovered

from FVW was assessed for their activity to use an equal enzyme units for the hydrolysis

of triglycerides. Candida cylindrical lipase showed the highest enzyme activity (21740

U/ gm of lipase) among all the lipases used followed by Candida rugosa lipase (10355

U/gm of lipase) while, Aspergillus niger (3391 U/gm), Thermomyces lanuginoca (4460

U/gm of lipase) and Mucor javanicus (3478 U/gm of lipase) showed lower activity

(Table 4.1).

Table 4.1. Properties of lipases used for the hydrolysis of fish oil during the processing

Source of lipase Specificity Activity (Units)

Aspergillus niger (AN) 1-,3->>2- 3391

Candida rugosa (CR) None 10355

Candida cylindrical (CC) None 21740

Thermomyces lanuginoca (TL) 1-,3->>2- 4460

Mucor javanicus (MJ) 1-,3->>2- 3478

These result demonstrate that the non specific lipases used in this study had much

higher activity compared to sn-1,3- specific lipases in hydrolyzing oil recovered from

FVW. All these lipases were used to concentrate PUFA during recovery of oil from

FVW.

Fatty acid composition of FVW-FO triglyceride recovered through solvent extraction,

lactic acid fermentation and enzymatic hydrolysis

Pediococcus acidilactici NCIM5368 and Fungal Protease-P-amano® (P Amano6;

60,000 U/g) were found to be the best lactic acid bacteria and protease for the recovery of

oil from FVW. Therefore they were used for the recovery of oil either by fermentation or


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enzymatic hydrolysis. Fatty acid composition (%) of triglyceride in FVW-FO recovered

through different process is shown in Table 4.2. Irrespective of the process used for the

recovery of oil from FVW, no change in n-3 PUFA profile was observed in triglyceride

of recovered oil. The major fatty acids in the SFA fraction of the oils included palmitic

(16:0) and stearic acid (18:0) whereas palmitoic (14:1) and oleic (C18:1) were the major

MUFA. Linoleic (18:2n-6) and linolenic acid (18:3n-3), arachidonic acid (20:4n-6),

eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) were the major

PUFA in the oil. Results demonstrate that lactic acid fermentation and enzymatic

hydrolysis of oil does not affect the triglyceride fatty acid composition compared to oil

recovered by solvent extraction.

Table 4.2. Fatty acid composition (%) of triglyceride fraction of FVW-FO recovered by

solvent extraction, lactic acid fermentation and enzymatic hydrolysis

SFA (%) MUFA (%) PUFA (%)

14:0 16:0 18:0 16:1 18:1 18:2n-6 18:3n-3 20:4n-6 20:5n-5 20:5n-5

Fresh 4.3 24.3 7.1 11.2 27.6 8.79 8.22 4.2 2.29 2.72

LAF 4.7 25.2 6.8 10.7 27.1 7.98 8.41 4.5 2.38 2.83

EH 4.1 24.8 6.6 11.4 28.2 8.82 8.78 4.1 2.32 2.70

LAF – Lactic acid fermentation, EH – Enzymatic hydrolysis, SFA – saturated fatty acids,

MUFA – monounsaturated fatty acids, PUFA – polyunsaturated fatty acids; Values are

mean of 5 experiments

Effect of different lipases during fermentation and enzymatic hydrolysis on oil recovery

Oil yield after lactic acid fermentation and enzymatic hydrolysis was calculated as

total oil yield and final oil yield (after removal of FFA). Oil yield on lipase assisted

hydrolysis during LAF of FVW using Pediococcus acidilactici NCIM5368 is shown in

Figure 4.1. There was no significant (p>0.05) difference in total oil yield among different

lipases used during fermentation of FVW using Pediococcus acidilactici NCIM5368.

However, Final oil yield was significantly (p<0.05) lower in case of AN (40.24 %) and

CC (29.8%) lipase followed by TL lipase (32.2%) added before LAF. Similarly, other

lipases assisted hydrolysis also slightly lowered the total oil yield compared to control.


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Acid value increased (2.4 - 5.8 fold) significantly (p<0.05) in the lipase treated oil

compared to control (Figure 4.2). Oil recovered on AN, TL and CC lipase hydrolysis

resulted in significantly (p<0.05) higher acid value compared other lipases (Figure 4.2).

Results on final oil yield and acid value demonstrate that higher degree of hydrolysis of

triglyceride with AN, TL and CC lipase compared to CR and MJ.

0

15

30

45

60

75

90

105

Control AN CR CC TL MJ

Tot

al o

il yi

eld

/ F

inal

oil

yiel

d (

%) Total oil yield Final oil yield

Source of lipases

aa

aa a aa

bb b,c

c c

Figure 4.1. Percent of total and final oil yield on lipase hydrolysis during oil recovery by

lactic acid fermentation of FVW using Pediococcus acidilactici NCIM5368,Values are

mean ± SD (n=5), Values not sharing common alphabet for the same pattern are

significantly different (p<0.05).


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Figure 4.2. Effect of lipases added during lactic acid fermentation of FVW on acid value

(mg KOH required to nutralise FFA) of recovered oil. Values are mean ± SD (n=5),

Values not sharing common alphabet for the same pattern are significantly different

(p<0.05)

In case of enzymatic hydrolysis, similar to lactic acid fermentation there was no

significant (p>0.05) difference in total oil yield when different lipases were used (Figure

4.3). Final oil yield was significantly (p<0.05) lower in comparison to control in lipase

aided enzymatic hydrolysis. The extent of decrease in final oil yield was lower (10.18 –

22.8 %) in enzymatic hydrolysis compared to lactic acid fermentation (21.6 – 40.24 %).

Acid value increased (0.73 - 1.9 fold) significantly (p<0.05) in the lipase treated oil

compared to control. Acid value was higher in case of AN (1.9 fold), TL (1.36 fold) and

CC (1.64 fold) lipases compared to other lipase which followed a similar trend as lactic

acid fermentation (Figure 4.4). The increase in acid value was lower (0.73 - 1.9 fold) in

enzymatic hydrolysis compared to lactic acid fermentation (2.4 - 5.8 fold) which may be

due to lower hydrolysis of triglyceride.


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0

20

40

60

80

100

Control AR CR CC TL MJ

Total oil yield Final oil yield

Source of lipases

Tot

al o

il yi

eld/

Fin

al o

il yi

eld

(%)

a

bb

a aaaa

bb

ba

Figure 4.3. Effect of lipases on total and final oil yield during oil recovery by enzymatic

hydrolysis of FVW using Fungal protease; Values are mean ± SD (n=5),

Values not sharing common alphabet for the same pattern are significantly

different (p<0.05)

Figure 4.4. Effect of lipases added during enzymatic hydrolysis (Fungal proteases) on

acid value (mg of KOH required to neutralize FFA) of recovered oil. Control

– no added lipase; Value are mean ± SD (n=5); Value not sharing common

alphabets for same pattern are significantly different.


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Triglyceride fatty acid composition after hydrolysis

Hydrolysis of FVW triglyceride by lipases caused varying degree of changes in

triglyceride fatty acid composition (Table 4.3). Ideally, enzymatic hydrolysis (lipase)

results in FFA removal that exhibits an increase in EPA and DHA and decrease in SFA

and MUFA concentration. Table 4.3 shows changes in 16:0, 18:0, 16:1, 18:1, 18:3n-3,

20:5n-3 and 22:6n-3 content in the final product obtained after lipase catalyzed

hydrolysis during lactic acid fermentation followed by FFA removal. Among the lipase

used during lactic acid fermentation, hydrolysis of oil with AN lipase resulted in higher

concentration of PUFA (39.7%) compared to control (26.5 %). In specific, hydrolysis

with AN lipase during LAF also resulted in higher concentration of DHA (5.7%)

compared to control (3.1 %). Further, the EPA (46.2%) and ALA (49.2%) content also

increased significantly (p<0.05) in AN hydrolyzed oil compared to control. Among the

lipases used, MM lipase showed the lowest effect on n-3 PUFA concentration. This study

reveals that hydrolysis with sn-1,3 specific lipase AN and TA followed by a non specific

lipase CC resulted in successful concentration of EPA+DHA in FVW-FO.

Effect of lipase on fatty acid composition (%) of lipid recovered on enzymatic

hydrolysis is shown in Table 4.4. Similar to fish oil recovered by LAF, there was increase

in PUFA content which ranged from 3.9 – 25.3% and was higher in AR (25.3 %)

followed by TL (21.4%) and CC (19.1%) lipase treated oil. Extent of concentration of

PUFA was lower in case of lipase added during enzymatic hydrolysis which correlates

with acid value of recovered oil.


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Table 4.3. Fatty acid composition (%) of triglyceride fraction of lipase treated oil

recovered on lactic acid fermentation of FVW.

Fatty acid Control A niger C rugosa C cylindrical T laniginosa M javanicus

Saturated Fatty acids (SFA)

14:0 2.8±0.5a 2.1±0.6a 2.1±0.7a 2.5±0.4a 2.4±0.5a 2.6±0.4a

15:0 1.8±0.3a 1.3±0.3a 1.4±0.2a 1.4±0.3a 1.4±0.2a 1.7±0.3a

16:0 29.7±2.8a 20.6±2.3b 22.6±3.1b 22.0±2.5b 22.7±2.4b 25.7±2.6a.b

17:0 1.7±0.2a 1.4±0.2 a 1.5±0.3a 1.5±0.2a 1.4±0.2a 1.5±0.3a

18:0 5.4±0.6a 3.2±0.3b 4.3±0.5a,c 4.7±0.7a.c 3.8±0.5b,c 4.8±0.5a

ΣSFA 41.4±4.1a 28.6±3.2b 31.9±3.3b 32.1±3.8b 31.7±4.2b 36.3±4.5a,b

Monounsaturated fatty acid (MUFA)

16:1 5.9±0.4a 4.9±0.6a 5.0±0.8a 5.3±0.4a 5.2±0.6a 5.4±0.8a

17:1 0.9±0.1a 0.7±0.1a 0.8±0.2a 0.9±0.1a 0.7±0.1a 0.7±0.2a

18:1n-9 17.8±1.3a 13.4±1.2b 17.2±1.6a 15.6±1.4 a,b 15.7±1.1 a,b 17.0±1.3a

18:1n-7 3.8±0.3a 2.7±0.3 b 3.2±0.3a,b 3.2±0.4a,b 3.6±0.2a 3.5±0.3 a

ΣMUFA 28.4±2.8a 21.7±2.6b 26.2±2.5a,b 25.0±2.6a,b 25.2±2.4a,b 26.6±2.4a,b

Polyunsaturated fatty acids (PUFA)

18:2n-6 10.9±1.3a 14.2±1.2b 12.5±1.1a 12.6±1.0a 12.7±1.4a 11.1±0.3a

18:3n-3 8.7±1.4a 13.0±1.2b 11.0±0.6c 10.7±0.6a,c 10.8±0.4c 10.2±1.3a,c

20:4n-6 1.3±0.4a 2.9±0.6 b 1.7±0.4 a 2.3±0.5 b 2.7±0.4b 1.5±0.3a

20:5n-3 2.6±0.4a 3.8±0.3 b 3.0±0.4 a 3.3±0.4a,b 3.4±0.4a,b 2.8±0.3a

22:6n-3 3.1±0.6a 5.7±0.8 b 3.9±0.7 a,c 4.5±0.6 b,c 4.8±0.5 b,c 3.3±0.4a

ΣPUFA 26.5±2.4a 39.6±2.2b 32.1±2.5c 33.4±2.2 c 34.4±2.9 c 28.9±2.0a

ND – not detected; Values are mean ± SD, Values not sharing common alphabet for the

same row are significantly different (p<0.05).


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Table 4.4. Fatty acid composition (%) of triglyceride fraction of lipase treated oil

recovered by proteolytic hydrolysis of FVW.

Fatty acid Control A niger C rugosa C cylindrical T laniginosa M javanicus

Saturated Fatty acids (SFA)

14:0 3.5±1.2a 2.4±0.9a 3.3±1.0a 2.6±0.8a 2.5±0.9a 2.0±1.1a

15:0 1.5±0.3 a 1.3±0.2a 1.2±0.2a 1.2±0.3a 1.1±0.2a 1.4±0.2a

16:0 29.1±2.2 a 25.6±3.1a 27.5±2.3a 27.0±2.7a 28.9±2.4a 28.7±2.2a

17:0 1.4±0.2 a 1.2±0.2a 1.3±0.1a 1.2±0.3a 1.3±0.2a 1.3±0.3a

18:0 4.8±0.6 a 3.9±0.4a 4.2±0.3a 4.1±0.5a 4.0±0.3a 4.6±0.4a

ΣSFA 40.3±2.9 a 34.4±2.7b 37.5±4.2a,b 36.1±3.6a,b 37.8±3.3a,b 38.0±2.9a,b

Mono – unsaturated fatty acid (MUFA)

16:1 9.0±0.8 a 7.2±0.6b 8.7±0.8 a 8.6±0.7 a 8.2±0.6 a,b 8.7±0.6 a

17:1 1.0±0.1a 0.8±0.1a 0.9±0.1 a 0.8±0.1 a 0.8±0.1 a 1.0±0.2 a

18:1n-9 18.9±1.7 a 16.1±1.4a 17.7±1.5 a 16.6±1.7 a 16.9±1.3 a 18.8±1.6 a

18:1n-7 2.9±0.3 a 2.1±0.3b 2.7±0.3 a,b 2.8±0.2a 2.7±0.2a 2.8±0.3a

ΣMUFA 31.8±2.2 a 26.2±2.1b 30.0±2.4a,b 28.8±2.6 a,b 28.6±2.7 a,b 31.3±2.0 a

Polyunsaturated fatty acids (PUFA)

18:2n-6 10.6±1.1a 12.6±0.9 a 12.2±0.8 a 12.1±1.3 a 12.4±1.2 a 11.1±1.0 a

18:3n-3 8.2±0.7a 10.3±0.8 b 9.3±0.9 a,b 9.6±1.2 a,b 9.7±0.9 a,b 8.4±1.1 a,b

20:4n-6 1.3±0.3a 2.1±0.4b 1.7±0.2a,b 2.0±0.3b 1.9±0.3b,a 1.3±0.2a

20:5n-3 2.6±0.3a 3.3±0.3b 3.1±0.3a,b 3.2±0.4a,b 3.4±0.3b 2.6±0.2a

22:6n-3 3.0±0.3a 3.9±0.4b 3.4±0.4 a,b 3.7±0.5a,b 3.8±0.4b 2.8±0.3a

ΣPUFA 25.7±2.3a 32.2±3.5b. 29.7±2.6a,b 30.6±2.7a,b 31.2±3.4b 26.3±2.6a,b

ND – not detected; Values are mean ± SD, Values not sharing common alphabet for the

same row are significantly different (p<0.05).

Comparing the fermentation and enzymatic hydrolysis methods, lipase aided LAF

has shown to be more effective in concentrating PUFA in recovered fish oil. However, in

both the methods, AN lipase resulted in maximum concentration of PUFA. The decrease


0

in SFA and MUFA content on AN lipase treatment during LAF was 30.9 and 23.6 %

respectively whereas during EH was by 14.6 and 17.1% respectively.

Time course study of hydrolysis with Aspergillus niger lipase during lactic acid

fermentation and enzymatic hydrolysis of lipids

As sn-1,3 specific lipase from Aspergillus niger showed better concentration

/enrichment of PUFA compared to other lipases used, effect of hydrolysis time on the

concentration of EPA + DHA in triglyceride was studied. Concentration of EPA and

DHA in triglyceride was estimated during fermentation (at the interval of 4 h till 24 h)

and enzymatic hydrolysis (at the interval of 15 min till 120 min) as shown in Table 4.5.

Table 4.5. Experimental plan for the effect of time on lipase hydrolysis of fish oil during

lactic acid fermentation.

Fermentation

Lipase

Addition

Fermentation + Lipase hydrolysis

Fermentation Time (h) 0 24 28 32 36 40 44 48

Lipase hydrolysis time (h) ___ 0 4 8 12 16 20 24

Figure 4.5. Effect of time (hours) on concentration of EPA and DHA in acylglycerol on

lipase hydrolysis during lactic acid fermentation; Values are mean ± SD, Values

not sharing common alphabet for the same pattern are significantly different

(p<0.05).


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Results show that there was significant increase (p<0.05) in both EPA (2.5 – 3.8

%) and DHA (3.1 – 5.7%) till 16th hour of hydrolysis with no increase afterwards,

suggesting 16 hours of reaction time during LAF may be sufficient enough to obtain

higher levels of EPA and DHA in the final product. There was no change in EPA and

DHA content in control (no lipase) compared to oil recovered by solvent extraction.

After 40 h of LAF (16 h lipase hydrolysis) (Table 4.5), SFA and MUFA content

reduced significantly (p<0.05) whereas there was increase in PUFA content (49.4 %) in

the oil compared to control (Figure 4.6). The concentration of PUFA is may be due to the

hydrolysis of SFA and MUFA in the sn-1,3 position of the triglyceride in the FVW-FO

during LAF and their removal.

0

5

10

15

20

25

30

35

40

45

50

SFA MUFA PUFA n3 PUFA

Control

AR Lipase

%

a

b

a

aa

b

b

b

Figure 4.6. Effect of Aspergillus niger Lipase (16th hour) during lactic acid fermentation

on concentration of SFA (saturated fatty acids), MUFA (monounsaturated fatty acids),

PUFA (polyunsaturated fatty acids) and n-3 PUFA Values are mean ± SD, Values not

sharing common alphabet for the same group of fatty acids are significantly different

(p<0.05).

Effect of time on hydrolysis of fish oil during enzymatic hydrolysis (fungal

protease) was optimized for maximum concentration of PUFA (Table 4.6). In case of

enzymatic hydrolysis, there was consistent and slow increase in EPA and DHA content

till 120th min (180 min of EH) as lipase was added after 60 min of hydrolysis (Figure

4.7). The increase in EPA and DHA during EH on lipase addition was lower compared to


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LAF of FVW. There was significant (p<0.05) change in SFA, MUFA and PUFA content

in the final oil (Figure 4.8) but the decrease was lower compared to oil recovered by

LAF. Results on concentration/enrichment of PUFA suggest that lipase addition during

LAF can be a better approach to improve PUFA levels in recovered fish oil.

Table 4.6. Experimental plan showing the effect of time on lipase hydrolysis of fish oil

during enzymatic hydrolysis (fungal protease).

EH

Lipase

Addition

EH + Lipase hydrolysis

EH Time (min) 0 60 75 90 120 150 180

Lipase hydrolysis time (min) ___ 0 15 30 60 90 120

EH – enzymatic hydrolysis with fungal protease

Figure 4.7. Effect of time (min) on concentration of EPA and DHA in acylglycerol on

lipase hydrolysis during enzymatic hydrolysis (EH).

The comparison of lipase hydrolysis during lactic acid fermentation and enzymatic

hydrolysis of EPA, DHA and PUFA level in the final oil is given in Table 4.7. Results

suggests that lipase hydrolysis during lactic acid fermentation is a better method

compared to proteolytic hydrolysis for the enrichment of PUFA during the recovery of

lipids.


Page 113

0

5

10

15

20

25

30

35

40

45

SFA MUFA PUFA n-3 PUFA

Control AR Lipase

%

a

ba

a

ab

b

b

Figure 4.8. Effect of Aspergillus niger lipase hydrolysis during enzymatic hydrolysis

(Fungal protease) on concentration of SFA (saturated fatty acids), MUFA

(monounsaturated fatty acids), PUFA (polyunsaturated fatty acids) and n-3 PUFA;

Values are mean ± SD, Values not sharing common alphabet for the same group of fatty

acids are significantly different (p<0.05).

Table 4.7. Comparison of PUFA enrichment during lactic acid fermentation and

enzymatic hydrolysis

Process EPA DHA PUFA

Solvent extracted 2.5±0.2a 3.1±0.2a 26.3±2.4a

LAF 2.7±0.2a 3.0±0.4a 26.4±3.1a

EH 2.6±0.2a 3.1±0.2a 25.7±2.6a

LAF + AN lipase 3.9±0.3b 5.8±0.4b 39.7±4.1b

EH + AN lipase 3.3±0.3c 3.9±0.4c 32.2±2.3c

LAF – lactic acid fermentation, EH – enzymatic hydrolysis, PUFA – polyunsaturated

fatty acid, AN – Aspergilus niger, Values are mean ± SD, Values not sharing common

alphabet for the same column are significantly different (p<0.05)


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Effect of antioxidants on oxidation of FO recovered during lactic acid fermentation

Different antioxidants (α-tocopherol, BHT, BHA, TBHQ at 100ppm) were to

prevent the auto-oxidation of PUFA during the recovery of FVW-FO. The data presented

in Figure 4.9 reflects that antioxidants had a significant (p<0.05) effect on the formation

of oxidation products in the sample. Results show a lower PV value in antioxidant added

FVW compared to control FVW (no antioxidant added). The PV value (meq of O2/kg of

oil) of control fermented sample was 37.8 which reduced significantly (p<0.05) in the

BHT (40.9%), BHA (46.4 %) TBHQ (71.5 %) and tocopherol (63.1 %) added samples.

The lowest PV (meq of O2/kg of oil) value was found in TBHQ (10.8) added FVW

followed by α-tocopherol (13.5) added indicating superior antioxidant properties of

antioxidant used in that order.

Figure 4.9. Effect of antioxidants on peroxide value (PV) and acid value of oil recovered

on fermentation of FVW with Pediococcus acidilactici NCIM5368. BHT - butylated

hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ tertiary butyl hydroquinone

(TBHQ), Control – without antioxidant, Acid value – expressed as mg KOH required to

nutralize FFA; Values are mean ± SD (n=5) (n=5), Values not sharing common alphabet

for the same pattern are significantly different (p<0.05)

In case of acid value, there was no significant (p>0.05) difference among lipids

recovered on addition of antioxidants to homogenized FVW (Figure 4.9). Fatty acid

composition (%) of lipid recovered on fermentation with added antioxidant did not show


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any change in fatty acid composition (Table 4.8). In particular, concentration of PUFA

remained unchanged irrespective of the antioxidant added. As TBHQ and α-tocopherol

were the two potent antioxidant found to reduce the peroxide value without affecting the

acid value and fatty acid composition, hence they were used for further study.

Table 4.8. Effect of antioxidants on fatty acid composition of fish oil recovered by lactic

acid fermentation of fish visceral waste.

Fatty acids Tocopherol BHT BHA TBHQ Fresh Control

14:0 1.8±0.2 a 1.7±0.2 a 1.8±0.2 a 1.8±0.3 a 1.9±0.3 a 1.7±0.2 a

15:0 0.7±0.1 a 0.7±0.2 a 0.7±0.1 a 0.7±0.1 a 0.8±0.2 a 0.7±0.1 a

16:0 26.5±2.8 a 26.3±3.0 a 26.5±3.6a 28.1±2.5 a 27.2±2.9a 25.8±3.2 a

17:0 1.4±0.3 a 1.4±0.2 a 1.4±0.2 a 1.4±0.3 a 1.3±0.3 a 1.1±0.2 a

18:0 6.5±0.4 a 6.5±1.1 a 6.5±0.6 a 6.2±0.9 a 6.4±0.4 a 6.2±0.7 a

16:1 5.4±0.7 a 5.6±0.5 a 5.8±0.8 a 5.7±0.7 a 5.1±0.9 a 5.4±0.4 a

17:1 0.7±0.2 a 0.6±0.2 a 0.7±0.2 a 0.7±0.1 a 0.8±0.1 a 0.8±0.2 a

18:1n-9 25.0±3.4 a 25.0±2.7 a 24.8±2.5a 25.0±2.8 a 24.8±3.2a 24.3±3.0 a

18:1n-7 2.3±0.3 a 2.2±0.2 a 2.3±0.3 a 1.8±0.3 a 1.9±0.4 a 2.1±0.3 a

18:2n-6 10.2±0.9 a 9.1±1.1 a 9.2±0.8 a 8.6±1.6 a 8.8±1.4 a 9.4±0.8 a

18:3n-3 8.8±1.0 a 8.6±0.7 a 8.0±0.6 a 8.5±0.8 a 8.4±1.2 a 7.8±0.9 a

20:4n-6 1.1±0.3 a 1.2±0.3 a 1.6±0.4 a 1.2±0.2 a 1.1±0.2 a 1.6±0.4 a

20:5n-3 2.4±0.2 a 2.4±0.2 a 2.3±0.3 a 2.4±0.3 a 2.4±0.2 a 2.2±0.3a

22:6n-3 2.7±0.3 a 2.6±0.3 a 2.6±0.2 a 2.7±0.2 a 2.6±0.3 a 2.4±0.2a

BHT - butylated hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ tertiary butyl

hydroquinone (TBHQ), Fresh – oil recovered by solvent extraction, control – LAF

without antioxidant; Values are mean ± SD (n=5), Values not sharing common alphabet

for the same pattern are significantly different (p<0.05)

As TBHQ and α-tocopherol were the better antioxidant, experiment for

optimization of their minimum concentration to prevent oxidation of PUFA was carried

out. LAF was carried out at different concentration (50, 100, 150 and 200ppm) of

selected antioxidant. Among the antioxidant used, TBHQ (68.5 %) and α-tocopherol


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(55.2%) showed significant (p<0.05) reduction in PV at lowest concentration of 50 ppm.

TBHQ and α-tocopherol showed the maximum reduction of 100% at 100 ppm and 150

ppm respectively, beyond which there was no change in peroxide value. The values were

similar to the peroxide values of the freshly extracted fish oil by solvent extraction

(Figure 4.10).

0

5

10

15

20

25

30

35

40

50 100 150 200 Fresh Control

TBHQ α-Tocopherol

Concentration in ppm

mg

of O

2/kg

oil

a cb

bb

a

bb

dc

cc

Figure 4.10. Effect of different concentration of TBHQ (tertiary butyl hydroquinone) and

α-tocopherol on peroxide value (meq of O2/kg) of oil recovered by lactic

acid fermentation. Fresh – oil recovered by solvent extraction, control –

without antioxidant; Values are mean ± SD (n=5), Values not sharing

common alphabet for the same pattern are significantly different (p<0.05)

Studies on the effect of antioxidants added to FVW before lactic acid fermentation

by Pediococcus acidilactici NCIM5368 suggests that addition of 100 ppm of TBHQ or

150 ppm of α-tocopherol to FVW can prevent the oxidation of PUFA during the

fermentation process of FVW.


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Effect of antioxidants on oxidation of FO recovered during enzymatic hydrolysis

The effect of TBHQ, BHT, BHA and α-tocopherol added to homogenized FVW

before initiation of enzymatic hydrolysis showed a significant (p<0.05) reduction in the

formation of oxidation product in the recovered fish oil. The PV value was lower in oil

recovered from antioxidant added FVW (before hydrolysed with Fungal protease)

compared to control (no antioxidant). The PV value of control oil was 37.5 meq O2/kg of

which reduced significantly (p>0.05) in tocopherol (66.7%), TBHQ (75.7%), BHT

(50.5%) and BHA (58.5 %) added groups (Figure 4.11). The lowest PV value (meq O2/kg

of oil) in fish oil stabilized with TBHQ (9.1) and α-tocopherol (12.5) at 100ppm

concentration demonstrating that these antioxidants are the best for the stabilization of

FVW-FO.

0

5

10

15

20

25

30

35

40

45

α-Tocopherol BHT BHA TBHQ Fresh Control

Acid value Peroxide value

Antioxidants

Aci

d /

Per

oxid

e va

lue

a

b

a aaaaa

c

d d

e

Figure 4.11. Effect of antioxidants on peroxide value (meq of O2/kg of oil) and acid

value (mg KOH/ kg of oil) of oil recovered on enzymatic hydrolysis of FVW with Fungal

proteases. BHT - butylated hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ -

tertiary butyl hydroquinone, Fresh – oil recovered by solvent extraction, control –without

antioxidant. ; Values are mean ± SD (n=5), Values not sharing common alphabet for the

same pattern are significantly different (p<0.05)

There was no significant (p>0.05) change in the acid value of fish oil recovered

from FVW by enzymatic hydrolysis with added antioxidants. However, acid value of FO-


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EH was non-significantly higher in case of BHT (11.9 %) and BHA (10.8 %) added

FVW. Effect of α-tocopherol, TBHQ, BHT and BHA on fatty acid composition of FVW-

FO is shown in Table 4.9. Addition of antioxidants to FVW before enzymatic hydrolysis

did not change the fatty acid composition of the recovered oil compared to that of

recovered by solvent extraction. In particular, concentration of n-3 PUFA (EPA, DHA

and ALA) remained unchanged irrespective of antioxidant added (Table 4.8).

Table 4.9. Effect of antioxidants on fatty acid composition (%) of fish oil recovered by

enzymatic hydrolysis of fish visceral waste

Fatty acids α-Tocopherol BHT BHA TBHQ Fresh Control

14:0 1.8±0.2a 1.7±0.1a 1.8±0.2a 1.8±0.2a 1.9±0.2a 1.7±0.2a

15:0 0.7±0.2a 0.7±0.8a 0.7±0.1a 0.7±0.1a 0.8±0.2a 0.7±0.1 a

16:0 26.5±2.8a 26.3±2.5a 26.5±2.6a 28.1±3.1a 27.2±2.4a 25.8±3.0a

17:0 1.4±0.3a 1.3±0.2a 1.4±0.2a 1.4±0.3a 1.3±0.2a 1.1±0.2a

18:0 6.5±0.4a 6.6±0.7a 6.5±1.1a 6.2±0.9a 6.4±0.8a 6.2±0.5a

16:1 5.4±0.5a 5.6±0.8a 5.8±0.6a 5.7±0.7a 5.1±0.4a 5.4±0.6a

17:1 0.7±0.1a 0.6±0.2a 0.7±0.2a 0.7±0.1a 0.8±0.1a 0.8±0.1a

18:1n-9 25.0±3.2a 25.0±2.8a 24.8±2.9a 25.0±2.7a 24.8±3.1a 24.3±2.6a

18:1n-7 2.3±0.2a 2.2±0.2a 2.3±0.3a 1.6±0.3a 1.9±0.2a 2.1±0.2a

18:2n-6 10.2±1.6a 9.1±0.7a 9.2±1.2a 8.6±1.0a 8.8±0.7a 9.4±0.5a

18:3n-3 8.2±0.5a 8.3±0.9a 8.0±0.4a 8.4±0.6a 8.2±0.7a 7.9±0.7a

20:4n-6 2.4±0.2a 2.5±0.3a 2.6±0.3a 2.4±0.3a 2.5±0.3a 2.6±0.2a

20:5n-3 2.3±0.2a 2.2±0.2a 2.2±0.1a 2.3±0.3a 2.3±0.2a 2.1±0.2a

22:6n-3 2.8±0.2a 2.7±0.2a 2.6±0.3a 2.8±0.3a 2.8±0.2a 2.6±0.2a

BHT - butylated hydroxytoluene, BHA - butylated hydroxyanisole, TBHQ tertiary butyl

hydroquinone, Control – without antioxidant. Fresh – oil recovered by solvent extraction,

control – enzymatic hydrolysis without antioxidant; Values are mean ± SD (n=5), Values

not sharing common alphabet for the same pattern are significantly different (p<0.05)

TBHQ and α-tocopherol were found to be the better antioxidants in preventing

oxidation of PUFA during the recovery of FVW-FO. Hence, they were chosen for further

studies to find out their concentration dependent effect on oxidation. EH of FVW was


Page 119

carried out with 50, 100, 150 and 200ppm of the selected antioxidant. PV of control oil

recovered by enzymatic hydrolysis of FVW was 36.5 meq of O2/kg of oil which reduced

significantly (p<0.05) on addition of TBHQ (50 ppm) and α-tocopherol (50 ppm) (Figure

4.12). Concentration of TBHQ and α-tocopherol required to mentain the PV (reduce the

auto-oxidation) similar to fresh (extracted by solvent) in 100 ppm and 150 ppm

respectively.

0

5

10

15

20

25

30

35

40

45

50 100 150 200 Fresh Control

TBHQ α-Tocopherol

Concentration in ppm

meq

of

O2/

kg o

f oi

l

aa

b

dc

bbb

b ccc

Figure 4.12. Effect of concentration of TBHQ and α-tocopherol on peroxide value (meq

of O2/kg of oil) of oil recovered by enzymatic hydrolysis. TBHQ - tertiary

butyl hydroquinone. Fresh – oil recovered by solvent extraction, control –

LAF without antioxidant; Values are mean ± SD (n=5), Values not sharing

common alphabet for the same pattern are significantly different (p<0.05)

In general effect of TBHQ on PV and acid value during LAF and EH of FVW

was found to be similar. The results further demonstrate that TBHQ is a better

antioxidant compared to other antioxidant used. The results conclude that 100 ppm of

TBHQ or 150 ppm of α-tocopherol can be recommended to reduce the oxidation of

recovered fish oil during LAF or EH of FVW. These antioxidants can be added to

homogenized FVW at the recommended concentration to stabilize PUFA during the

recovery of fish oil from FVW.


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Discussion

Enrichment/concentration of PUFA using lipases in recovered oil

In the previous chapter standardization of lactic acid fermentation and enzymatic

hydrolysis for the recovery of lipids from FVW has been carried out. Fatty acid

composition of FO-LAF and FO-EH showed equal distribution of SFA, MUFA and

PUFA. PUFA concentrates in triglycerides, devoid of more saturated fatty acids, are

much better than original oil themselves because they allowed daily intake of total lipid

to be as low as possible.

Lipase aided hydrolysis during recovery of FVW-FO caused changes in acid value

and fatty acid composition at varying levels. Higher concentration of PUFA in

triglyceride was observed in case of lipase aided hydrolysis during lactic acid

fermentation compared to proteolytic enzymatic hydrolysis. Lipase catalyzed hydrolysis,

especially with AN and TL sn-1,3-specific lipases resulted in successful concentration of

EPA and DHA with different efficiencies. Ideally, enzymatic hydrolysis using lipases

followed by removal FFA increases the EPA and DHA concentrations by reducing SFA

and MUFA. Aspergillus niger sn-1,3-specific lipase used in the study was significantly

higher compared to other lipases in concentrating PUFA during the recovery process.

Okada and Morrissey (2007) have reported that non-specific lipases was better than sn-1,

3-specific lipase in concentrating PUFA in sardine oil. In case of fish oil, the second

position of the glycerol moiety is usually more enriched with n-3 PUFAs (Bornscheuer,

2000), although this may vary depending on species (Gamez-Meza et al. 1999). The

results on concentration of PUFA by sn-1,3 specific lipase also highlights the presence of

n-3 PUFA on the sn-2 position in FVW-FO.

Concentration of PUFA was also observed in the non-specific lipase CC in this

study. This indicates the ability of CC lipases to discriminate SFAs and MUFAs from n-3

PUFA in FO-FVW, most likely due to the reduced steric hindrance observed with SFAs

and MUFAs when linked to a glycerol backbone (Gamez-Meza et al. 2003). The

molecular conformation of cis carbon–carbon double bonds in PUFAs, particularly EPA

and DHA, causes steric hindrance and subsequent bending of the fatty acid chains,

bringing the terminal methyl groups very close to the ester bonds (Chakraborty et al.

2010). Because of this steric hindrance effect, enzymatic active sites cannot reach the


Page 121

ester-linkages of these fatty acids with their glycerol backbones, thereby protecting EPA

and DHA from lipase-catalyzed hydrolysis. However, this does not occur with the

relatively straight chains of SFAs and MUFAs, and therefore hydrolysis is not hindered

(Shahidi and Wanasundara, 1998; Carvalho et al. 2002). Also, it has been suggested that

TGs without EPA and DHA are hydrolyzed in the first phase, and TGs with EPA and

DHA are hydrolyzed later, indicating that the lipase recognizes the whole molecular

structure, not only its ester bonds (Hoshino et al. 1990).

Lipase catalyzed hydrolysis was demonstrated to be a feasible method for

concentration of n-3 PUFAs during lactic acid fermentation and enzymatic hydrolysis.

Use of lipase to produce n-3 PUFA concentrates has an advantage over traditional

methods such as chromatographic separation, molecular distillation etc. of concentration

because such procedures involves extreme pH and high temperature which may affect the

quality of oil. Therefore, the mild conditions using enzymatic hydrolysis provide a

promising alternative that could also save energy and increase product selectivity. In

addition, the enzymatic hydrolysis method produces n-3 fatty acids in the glycerol form,

which is considered nutritionally favorable.

Stabilization of PUFA during fermentation and enzymatic hydrolysis

Due to the presence of multiple double bonds in PUFAs, they are highly susceptible

to oxidation and the oxidation products can have adverse health effects to the consumer

due to their cytotoxic and genotoxic effects (Esterbauer, 1993). Peroxide value (PV) is a

measure of the extent of oxidation of a lipid system. This value indicates the quantity of

oxidized substances, normally hydroperoxides, which liberate iodine from potassium

iodide under specified conditions (Rogers et al. 2001; Yanishlieva and Marinova, 2001).

Fish oil during lactic acid fermentation and enzymatic hydrolysis may decompose readily

which is measured by an increase in PV. In our present study, different antioxidants were

assessed find to find out their ability to protect PUFA from oxidation during the recovery

of FVW-FO. Results have shown that 100 ppm of TBHQ and 150 ppm of α-tocopherol

prevents auto-oxidation of fish oil during recovery of oil by lactic acid fermentation and

enzymatic hydrolysis.

Antioxidants function by inhibiting or interrupting the free radical mechanism of

lipid auto-oxidation. Antioxidants or phenolic substances function as free radical


Page 122

acceptors, thereby terminating oxidation at the initial step and also scavenging radicals

formed later, in the oxidation process (Wang et al.2011). The antioxidant and free radical

complex is stable and does not split into other compounds that provide off-flavor and

odors, nor does it propagate further oxidation of the lipid (O’Brien, 1998). In the present

study, TBHQ and α-tocopherol were found to be the best antioxidant compared to other

in reducing peroxide value. Previous studied by Haung et al (1994) with corn oil and by

Kulas and Ackman (2001) on fish oil showed 100 ppm α-tocopherol as a concentration

for maximal antioxidant activity in those oil.

It is concluded that TBHQ (100ppm) and α-tocopherol (150ppm) minimizes the

oxidation of fish oil during fermentation and enzymatic hydrolysis. These antioxidants

can be used to homogenized FVW before lactic acid fermentation/enzymatic hydrolysis

to recover good quality (unoxidized) lipids. In case of enrichment/ concentration of

PUFA by lipase treatment 1,3 specific AN lipase was the best among the lipase used in

our study for both LAF and EH.

chapter iv –enrichment and stabilization of...

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