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Inactivation of lipase enzyme by using chemicals to maximize rice bran shelf life and its edible oil recovery

Muhammad Akhter

Rice Research Institute, Kala Shah Kaku, Lahore, Pakistan

E-mail : director_rice@yahoo.com

Nazia Afzal

Government Fatima Jinnah College for Women, Lahore, Pakistan

Zulqurnan Haider

Rice Research Institute, Kala Shah Kaku, Lahore, Pakistan

Mohsin Ali Raza

Rice Research Institute, Kala Shah Kaku, Lahore, Pakistan

DOI: 10.15761/IFNM.1000123

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Abstract

Rice bran is the by-product of the rice milling industries so in view of growing need and consciousness about the nutritional and functional properties the rice bran is very important co-product. In this research article the methods were described regarding different chemicals with different concentration in controlling the activity of lipase enzyme and ultimately to maximize its oil recovery from the rice bran during 60 days of storage in a room temperature. The use of hydrochloric acid at concentration of about 30 ml/Kg helps greatly in controlling the lipase enzyme activity and reduces the % of FFA while the other chemicals used in this study (Phosphoric acid, Acetic acid, Sodium metabisulphite) failed to control in the rise of free fatty acid contents. The chemicals can be applied easily by sprinkling or spraying. This operation done on small rice bran lots through manual hand mixing. This method of chemical stabilization of rice bran is really a useful method in the rice mills where there is shortage of electricity or steaming facility.

Key words

stabilization, HCL, rice bran, lipase enzyme, oil contents, inactivation

Introduction

Rice is an important food as well as cash crop of Pakistan. It is also a major export commodity and earns about US $ 2.2 billion foreign exchange annually.There is more than 750 million metric tons production of rice paddy annually all around the world [1]. Rice is greatly consumed all over the world and used as a staple food product in different regions of the whole world due to its nutritional value [2].

The grain of the rice contain about 2 to 3% fat, and this fat portion is mainly concentrated in the embryo or in the germ and then in the outer layer of the seed [3]. During milling the germ and the bran powder layers detachedfrom the endosperm and this milling finally concentrate the fat portion into the residue generally said as “Rice Bran” [4]. Rice bran is massive rice milling industry discard produced during rice polishing and mainly used in animal feed. It constitutes about 7-8% of the whole paddy grainwhich contributes about more than 60 million tons per annum of the whole world production [1,5]. Rice bran has great potential to contribute to supply edible oil to the world and it ranges about 10-26% of the rice bran depending upon degree of milling, climatic conditions and variety [2,6,7].

Research in the last decades showed that rice bran is an outstanding source of vitamins, minerals, antioxidants, proteins, fats and dietary fibers [8]. Several problems of raw bran management have restricted production of edible grade rice bran oil. One of the problem is high lipase activity in the bran, which quickly hydrolyzes the fats of oil into free fatty acids [9]. To resolve this main problem the use of proper technique or deactivation method is very important.

To date, several studies have been conducted on stabilization techniques of rice bran and its oil [9,10,11]. Although a number of studies like microwave heating, ohmic heating, dry or moist heat treatment and little bit on pH lowering have been conducted for rice bran and its oil stabilization [12 ]. However, treatments with different chemicals have not been properly used for this purpose.The objective of this research work was to investigate the effect of using different chemicals for the stabilization of rice branon lipase enzyme, which ultimately leads to enhance the shelf life of rice bran and then maximize its oil contents.

Materials and methods

Laboratory grade Hydrochloric acid, Phosphoric acid, Acetic acid and Sodium metabisulphite were purchased from Merck, Germany and Hexane (Solvent for Extraction) from Sigma-Aldrich, Germany in this present research work.Freshly milled rice bran powder samples were collected from our local rice mills situated in areas of Muridke District Sheikhupura and Kamoke, District. Gujranwala.Stabilization of rice bran was carried out by spreading rice bran in a layer of 5 cm thick and the required amount different chemicals (Hydrochloric acid used @ a rate of 20, 30 & 35 ml/Kg, Phosphoric acid used @ 0.5, 1.0 & 1.5% per Kg, Acetic acid used @ 3, 5 & 7% per Kg and Sodium metabisulphite used at 1.5, 2.0 & 2.5% per Kg) were sprinkled onbran layers with different concentrations and mixed well by hand using protective clothing.Total 12 treatments were prepared with above mentioned acids or chemicals each with 3 treatments with different concentration. After completion of the treatments, all the rice bran samples were packed in locally made polyethylene bags. Then the packed rice bran samples were stored in a room temperature and analyzed it for free fatty acid % and oil extraction % after every 10 days interval upto 60 days of storage [13].

Analytical methods

Extraction of the oil contents of the samples were conducted on a laboratory scale Soxhlet extractor (Sayre et al., 1985). Analytical grade hexane was used and hexane temperature during extraction was about 60°C. Determination of free fatty acid (FFA) was done by alkalimetric titration (AOAC, 2006). The proximate analysis of chemically stabilized rice bran for moisture, protein, fats, crude fiber and ash contents were determined by [14-17].

Fatty acid composition of the treated rice bran samples

First from the four chemicals, we select the best treatment from each chemical on the basis of rice bran oil FFA% and then covert this crude oil into refined form. The best treatments selected for the fatty acid composition analysis are as follows:

  1. Hydrochloric Acid (HCL) @ 30 ml/Kg
  2. Phosphoric Acid @ 1.5% W/W
  3. Acetic Acid @ 7% W/W
  4. Sodium Metabisulphite @ 2% W/W

All the above treatments were analyzed for fatty acid analysis by using the method of [18] in which the methyl esters of fatty acids were separated with a GC-17A (Shimadzu Co., Japan) column equipped with HP-20M (25 m × 0.32 mm, 0.3 µm) at the following temperature program: initial temperature of 180°C (10 min hold) to 200°C at 4/min (2 min hold) and to 220°C at 4/min (12 min hold). Identification of fatty acid methyl esters was made by comparing their relatives retention times with that of known standard samples (linoleic acid, oleic acid, palmitic acid, stearic acid, arachidic acid, linolenic acid,eicosenoic acid, myristic acid and behanic acid) (Figure1-5).

Figure 1. FFA and oil % of the rice bran treated with phosphoric acid during storage

Figure 2. FFA and oil % of the rice bran treated with hydrochloric acid (HCL) during storage

Figure 3. FFA and oil % of the rice bran treated with acetic acid during storage

Figure 4. FFA and oil % of the rice bran treated with sodium metabisulphite during storage

Figure 5:

Statistical analysis

Analysis of variance of the data was computed using the Statistica computer program. The Least Significance Difference test at 5% level of significance was used to test the differences among mean values [19].

Results and discussions

Proximate analysis of rice bran

The results of analysis regarding moisture, protein, fats, crude fiber and ash contents/percentage of procured rice bran from rice processing mills and local Sheller’swere shown in Table 1. The rice bran used in this research work selected from local rice Sheller’s having good nutritional properties regarding proximate analysis with 11.2% moisture content, 16.87% protein contents, 20.20% rice bran fats, 8.29% crude fiber and 9.21% of ash contents. All these values were significantly different from proximate analysis of rice bran procured from rice processing mills. The oil contents of the rice bran vary according to the different chemical treatments and shown in Table 2 to 5. Acid stabilization appears to facilitate extraction of the crude oil from the rice bran. Outof these chemical treatments hydrochloric acid (HCL, Table 3) gave the best results in achieving maximum oil recovery that were in the range of (16.8-13.8%) in T1, (16.6-15.1%) & (16-14.7%) in T2 and T3 in 60 days storage respectively. The treatment with concentration of 30 ml/Kg of HCL gives the best optimum results in % of Crude oil recovery. While in other chemical treatments it ranges from (16.2-12.96%) in phosphoric acid, (15.94-14.2%) in acetic acid and (15.6-14.68%) in sodium metabisulphitein all three treatments respectively. Statistical analysis showed significant differences in the oil contents. The ranges of oil contents in the present work match the results of [9,20,21].

Table  1. Proximate analysis of oils of rice bran collected from different sources

Parameters

T1 (From Local Sheller’s)

T2 (From Rice Processing mills)

Moisture

11.22

11.60

Protein

16.87

16.60

Fats

20.20

19.15

Crude Fiber

8.29

7.99

Ash Contents

9.21

8.75

Values presented are averages of duplicate samples

Table 2. FFA and oil % of the rice bran treated with phosphoric acid during storage

Treatments

T1

T2

T3

Concentrations (%)

0.50%

1%

1.50%

Parameters

FFA%

OIL%

FFA%

OIL%

FFA%

OIL%

 

Storage days

0 Day

3.38g

15.24a

3.13g

16a

3.09g

16.2a

10 Days

18.05f

14.88b

8.46f

15.8b

7.76f

15.8b

20 Days

24.25e

13.94c

17.62e

15.6b

17.06e

15.4c

30 Days

30.69d

13.5d

32.4d

15c

28.2d

15.4c

40 Days

34.72c

13.4d

43c

14.2d

40.8c

14.6d

50 Days

42.58b

13.12e

45.8b

13.4e

44.69b

13.4e

60 Days

50.9a

12.96f

47.9a

13.1f

46.1a

13.2f

Table 3. FFA and oil % of the rice bran treated with hydrochloric acid (HCL) during storage

Treatments

T1

T2

T3

Concentrations (%)

20ml/Kg

30ml/Kg

35ml/Kg

Parameters

FFA%

OIL%

FFA%

OIL%

FFA%

OIL%

 

Storage days

0 Day

2.9f

16.8a

2.81f

16.6ab

2.88f

16a

10 Days

5.64e

15.9b

4.94e

16.2a

4.72e

15.7b

20 Days

5.8d

15.6c

5.5d

16ab

5.36d

15.4c

30 Days

6.48c

15.4c

5.92c

15.8a

5.43d

15.3d

40 Days

9.7b

14.8d

6.1b

15.4ab

5.64c

15.1e

50 Days

9.8b

14.2e

6.42a

15.2b

5.92b

14.8f

60 Days

10.01a

13.8f

6.47a

15.1b

6.56a

14.7f


Free fatty acid (FFA) status of rice bran

The free fatty acids contents (FFA %) of rice bran is shown in Table 2 to 5. From all the treatments again HCL gave the good results in controlling the activity of lipases enzyme and from the three concentrations of HCL, concentration of 30 ml/Kg appears to give the best results in enzyme deactivation that is in the range of (2.81-6.47%) in 60 days.While the other two concentration of HCL also give satisfactory performance regarding enzymes deactivation that is (2.9-10.01%) in 20 ml/Kg and (2.88-6.56%) in 35 ml/Kg respectively in 60 days.The other chemical treatments failed in controlling lipase activity these treatments lose their control right after 10 and 20 days of storage and breaks the recommended limit of less than 10% FFA. All the results shown in the table gave significant differences in the FFA contents of rice bran and its oil contents. Prabhakar and Venkatesh [9] finding of FFA% depicts the results of the present study of FFA%.

Table 4. Free fatty acids and oil % of the rice bran treated with acetic acid during storage

Treatments

T1

T2

T3

Concentrations (%)

3%

5%

7%

Parameters

FFA%

OIL%

FFA%

OIL%

FFA%

OIL%

 

Storage days

0 Day

4g

15.7a

3.81g

15.91a

3.88g

15.94a

10 Days

6.1f

15.5a

4.94f

15.85a

4.79f

15.86a

20 Days

9.59e

15.09b

9.47e

15.71b

9.02e

15.78a

30 Days

11.99d

14.78c

11.56d

15c

11.28d

15.4b

40 Days

14.95c

14.6cd

14.32c

14.8d

13.96c

14.9c

50 Days

15.65b

14.41de

14.95b

14.65e

14.24b

14.73cd

60 Days

16.07a

14.2e

15.37a

14.62e

14.95a

14.58d

Table 5. FFA and oil % of the rice bran treated with sodium metabisulphite during storage

Treatments

T1

T2

T3

Concentrations (%)

1.50%

2%

2.50%

Parameters

FFA %

FFA %

FFA %

OIL%

FFA %

OIL %

 

Storage days

0 Day

3.88g

15.4a

3.81g

15.4a

3.8g

15.6a

10 Days

4.94f

15.35a

5.47f

15.39a

5.16f

15.26ab

20 Days

6.67e

15.23a

6.61e

15.39a

6.42e

15.26ab

30 Days

7.93d

15b

7.52d

15.15b

7.77d

15.1ab

40 Days

8.57c

14.84bc

8.27c

14.98bc

8.56c

14.9b

50 Days

9.92b

14.78c

9.87b

14.86c

9.9b

14.8b

60 Days

10.78a

14.68c

10.53a

14.77c

10.54a

14.68ab

Fatty acid composition of the treated rice bran samples

Table 6 shows the fatty acid (FA) composition results of treated rice bran samples.The contents of total saturated fatty acids of all the treatments were in the range of 0.27-0.35 Myristic acid (C 14:0), 18.95-22.82 Palmitic acid (C 16:0), 1.43-1.55 Stearic (C 18:0),0.62-0.76 arachidic (20:0), 0.71-1.24 Ecosanoic (20:1) and 0.43-0.76 Behanic acid (22:0) respectively. The content of total saturated fatty acids of rice bran oil appeared more in samples treated with phosphoric acid @ 1.5% W/W that is about about (32.19%) after that in acetic acid @ 7% W/W (29%), then in sodium metabisulphite @ 2% W/W (26%) and atlast in HCL treated sample @ 30 ml/Kg (22%). While in case of unsaturated fatty acid, the percentage of unsaturated fatty acids of all the treatments were in the range of 43.10-45.00 Oleic acid (C 18:1), 23.81-31.67 Linoleic acid (C 18:2) and0.90-1.15 Linolenicacid (C 18:3) respectively. The content of unsaturated fatty acids of rice bran oil appeared more in samples treated with Hydrochloric acid @ 30 ml/Kg that is about (77%) after that in sodium metabisulphite @ 2% W/W (74%),  acetic acid @ 7% W/W (70%) and phosphoric acid @ 1.5% W/W (68%) respectively. So the conclusion is that the sample of rice bran treated with HCL gives the best results regarding unsaturated fatty acids. The above results reveals the findings of [22].

Table 6. Fatty acid composition of rice bran samples treated with four different chemicals

Fatty Acids

HCL @ 30ml/Kg

Phosphoric Acid @ 1.5% W/W

Acetic Acid @ 7% W/W

Sodium Metabisulphite @ 2% W/W

C 14:0

0.27

0.35

0.35

0.33

C 16:0

18.95

22.82

21.04

19.02

C 18:0

1.43

1.55

1.54

1.46

C 18:1

45.00

43.10

43.20

44.25

C 18:2

31.67

23.81

26.82

29.19

C 18:3

1.15

0.90

0.92

1.05

C 20:0

0.72

0.62

0.76

0.73

C 20:1

0.71

1.24

1.08

0.74

C 22:0

0.44

0.76

0.43

0.47

∑SAFFA

21.81

26.1

24.12

22.01

∑USFFA

78.53

69.05

72.02

75.23

C 14:0 Myristic Acid, C 16:0 Palmitic Acid, C 18:0 Stearic Acid, C 18:1 Oleic Acid, C 18:2 Linoleic Acid, C 18:3 Linolenic Acid, C 20:0 Arachidic Acid, C 20:1 Eicosanoic Acid, C 22:0 Behenic Acid.

Conclusions

The chemical methods for stabilization of the rice bran provides an answer to the problem of handling raw bran deterioration mainly in the developing countries with numerous small rice mills which lack adequate electricity. Out of all the chemicals used in this experiment hydrochloric acid @ 30 ml/Kg gives the best results in the stabilization of rice bran from lipase enzyme during the storage of 60 days at room temperature.

Acknowledgement

This study was supported by a grant from the Punjab Agriculture Research Board and awarded to Rice Research Institute, Kala Shah Kaku. (From Year 2010-2013).

References

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Editorial Information

Editor-in-Chief

Masayoshi Yamaguchi
Emory University School of Medicine

Article Type

Research Article

Publication history

Received: March 20, 2015
Accepted: April 23, 2015
Published: April 27, 2015

Copyright

©2015 Akhter M. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Akhter M, Afzal N, Haider Z, Raza MA (2015) Inactivation of lipase enzyme by using chemicals to maximize rice bran shelf life and its edible oil recovery. Integr Food Nutr Metab 2: DOI: 10.15761/IFNM.1000123

Corresponding author

Muhammad Akhter

Rice Research Institute, Kala Shah Kaku, Lahore, Pakistan.

E-mail : director_rice@yahoo.com

Table  1. Proximate analysis of oils of rice bran collected from different sources

Parameters

T1 (From Local Sheller’s)

T2 (From Rice Processing mills)

Moisture

11.22

11.60

Protein

16.87

16.60

Fats

20.20

19.15

Crude Fiber

8.29

7.99

Ash Contents

9.21

8.75

Values presented are averages of duplicate samples

Table 2. FFA and oil % of the rice bran treated with phosphoric acid during storage

Treatments

T1

T2

T3

Concentrations (%)

0.50%

1%

1.50%

Parameters

FFA%

OIL%

FFA%

OIL%

FFA%

OIL%

 

Storage days

0 Day

3.38g

15.24a

3.13g

16a

3.09g

16.2a

10 Days

18.05f

14.88b

8.46f

15.8b

7.76f

15.8b

20 Days

24.25e

13.94c

17.62e

15.6b

17.06e

15.4c

30 Days

30.69d

13.5d

32.4d

15c

28.2d

15.4c

40 Days

34.72c

13.4d

43c

14.2d

40.8c

14.6d

50 Days

42.58b

13.12e

45.8b

13.4e

44.69b

13.4e

60 Days

50.9a

12.96f

47.9a

13.1f

46.1a

13.2f

Table 3. FFA and oil % of the rice bran treated with hydrochloric acid (HCL) during storage

Treatments

T1

T2

T3

Concentrations (%)

20ml/Kg

30ml/Kg

35ml/Kg

Parameters

FFA%

OIL%

FFA%

OIL%

FFA%

OIL%

 

Storage days

0 Day

2.9f

16.8a

2.81f

16.6ab

2.88f

16a

10 Days

5.64e

15.9b

4.94e

16.2a

4.72e

15.7b

20 Days

5.8d

15.6c

5.5d

16ab

5.36d

15.4c

30 Days

6.48c

15.4c

5.92c

15.8a

5.43d

15.3d

40 Days

9.7b

14.8d

6.1b

15.4ab

5.64c

15.1e

50 Days

9.8b

14.2e

6.42a

15.2b

5.92b

14.8f

60 Days

10.01a

13.8f

6.47a

15.1b

6.56a

14.7f


Table 4. Free fatty acids and oil % of the rice bran treated with acetic acid during storage

Treatments

T1

T2

T3

Concentrations (%)

3%

5%

7%

Parameters

FFA%

OIL%

FFA%

OIL%

FFA%

OIL%

 

Storage days

0 Day

4g

15.7a

3.81g

15.91a

3.88g

15.94a

10 Days

6.1f

15.5a

4.94f

15.85a

4.79f

15.86a

20 Days

9.59e

15.09b

9.47e

15.71b

9.02e

15.78a

30 Days

11.99d

14.78c

11.56d

15c

11.28d

15.4b

40 Days

14.95c

14.6cd

14.32c

14.8d

13.96c

14.9c

50 Days

15.65b

14.41de

14.95b

14.65e

14.24b

14.73cd

60 Days

16.07a

14.2e

15.37a

14.62e

14.95a

14.58d

Table 5. FFA and oil % of the rice bran treated with sodium metabisulphite during storage

Treatments

T1

T2

T3

Concentrations (%)

1.50%

2%

2.50%

Parameters

FFA %

FFA %

FFA %

OIL%

FFA %

OIL %

 

Storage days

0 Day

3.88g

15.4a

3.81g

15.4a

3.8g

15.6a

10 Days

4.94f

15.35a

5.47f

15.39a

5.16f

15.26ab

20 Days

6.67e

15.23a

6.61e

15.39a

6.42e

15.26ab

30 Days

7.93d

15b

7.52d

15.15b

7.77d

15.1ab

40 Days

8.57c

14.84bc

8.27c

14.98bc

8.56c

14.9b

50 Days

9.92b

14.78c

9.87b

14.86c

9.9b

14.8b

60 Days

10.78a

14.68c

10.53a

14.77c

10.54a

14.68ab

Table 6. Fatty acid composition of rice bran samples treated with four different chemicals

Fatty Acids

HCL @ 30ml/Kg

Phosphoric Acid @ 1.5% W/W

Acetic Acid @ 7% W/W

Sodium Metabisulphite @ 2% W/W

C 14:0

0.27

0.35

0.35

0.33

C 16:0

18.95

22.82

21.04

19.02

C 18:0

1.43

1.55

1.54

1.46

C 18:1

45.00

43.10

43.20

44.25

C 18:2

31.67

23.81

26.82

29.19

C 18:3

1.15

0.90

0.92

1.05

C 20:0

0.72

0.62

0.76

0.73

C 20:1

0.71

1.24

1.08

0.74

C 22:0

0.44

0.76

0.43

0.47

∑SAFFA

21.81

26.1

24.12

22.01

∑USFFA

78.53

69.05

72.02

75.23

C 14:0 Myristic Acid, C 16:0 Palmitic Acid, C 18:0 Stearic Acid, C 18:1 Oleic Acid, C 18:2 Linoleic Acid, C 18:3 Linolenic Acid, C 20:0 Arachidic Acid, C 20:1 Eicosanoic Acid, C 22:0 Behenic Acid.

Figure 1. FFA and oil % of the rice bran treated with phosphoric acid during storage

Figure 2. FFA and oil % of the rice bran treated with hydrochloric acid (HCL) during storage

Figure 3. FFA and oil % of the rice bran treated with acetic acid during storage

Figure 4. FFA and oil % of the rice bran treated with sodium metabisulphite during storage

Figure 5: