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Effect of galangal essential oils on enzyme activity and nutrient digestibility in in vitro rumen fermentation

Dewi Ratih Ayu Daning1,2, Budi Prasetyo Widyobroto1, Chusnul Hanim1, Muhlisin1 and Lies Mira Yusiati1*

1 Faculty of Animal Science, Universitas Gadjah Mada, Jl. Fauna 3, Bulaksumur, Yogyakarta - 55281, Indonesia
* liesmira@ugm.ac.id
2 Department of Animal Science, Politeknik Pembangunan Pertanian Malang, Ministry of Agriculture, Jl. Dr. cipto 144 a, Bedali, Lawang 65200

Abstract

With bioactive ingredients, including cineol, phenol and alkaloids, galangal is an aromatic plant that ranks third in Indonesian production behind turmeric and ginger. In addition, galangal contains phenolic compounds that can help protect proteins in the rumen. This study aimed to evaluate the effect of giving galangal essential oil on enzyme activity (CMC-ase, protease and amylase) and nutrient digestibility in the rumen and post-rumen in vitro. The treatments were: control (no additive), galangal EO (30, 60, 120 無) and cineole (5 無). Rumen fluid batch cultures in vitro for 48 and 96 hours with a 60:40 forage:concentrate ratio were used to assess the effects of the treatments. Data obtained were analyzed using one-way ANOVA and continued by DMRT. Galangal EO dose linearly lowered the activities of carboxymethyl cellulose (CMC-ase), amylase and protease (p < 0.05). Furthermore, galangal EO treatment reduced rumen digestibility of crude protein (CP), organic matter (OM) and dry matter (DM) (p < 0.05). Meanwhile, galangal EO treatments enhanced post rumen, DM, OM and protein digestibility (p < 0.05). Therefore, we conclude that galangal EO as a feed additive may be a potential way to improve nutrient utilization in ruminants.

Keywords: galangal EO, nutrient digestibility, in vitro


Introduction

The protein requirement of dairy cows is not enough coming from microbial protein but requires quality feed protein resistant to rumen degradation (Schwab and Broderick 2017; Widyobroto et al 2007). Protein degradation involves protease enzymes produced by proteolytic bacteria in the rumen, namely Prevotella ruminicola (Jami et al 2014). Strategies to reduce protein degradation can be done using phenol compounds (Grazziotin et al 2020). Phenolic compounds such as tannins from Swietenia mahagoni (Basri et al 2021) and Nutmeg leaves (Canadianti et al 2020) can reduce protein digestibility in the rumen because its hydroxyl group can bind to feed proteins and enzymes (Fitriastuti et al 2019, Yusiati et al 2018). However, it should be noted that phenol can bind to cellulose, starch and minerals (Qin et al 2016). Therefore, it is necessary to evaluate the dosage of bioactive compounds without disturbing the digestibility of other essential nutrients for ruminants. Sources of phenolic compounds can be found in several aromatic plants (Simitzis 2017).

Galangal production reached 498.7 tons in 2020 (BPS, 2020). The part of the galangal plant Alpinia galangal that is often used is the rhizome (Ministry of Trade 2011). Galangal rhizome contains essential methyl cinnamate, cineol, camphor, pinene, galanin and eugenol (Raina and Abraham 2017). Research conducted by Prakatthagomol et al (2011) proved that a concentration of essential oil from galangal rhizome Alpinia galangal could inhibit the activity of both Gram-negative and Gram-positive bacteria. Furthermore, studies of rosemary leave with the main compound similar to galangal EO has been shown to reduce the abundance of proteolytic bacteria (Cobellis et al 2016). Based on its function and bioactive components, galangal essential oil can modify the rumen bacteria population and reduce protein degradation in the rumen. When crude protein degradation in the rumen is decreased, nitrogen use efficiency can be improved and N2O emissions reduced (Blach et al 2015). This study aims to determine the effect of the dose of galangal essential oil on enzyme activity and nutrient digestibility in the rumen and post-rumen.


Materials and methods

Galangal EO preparations and nutrient compositions

The galangal rhizome comes from local farmers in Boyolali Regency, Central Java. The plant taxonomy has been identified in the Purwodadi Botanical Gardens, National Research and Innovation Agency (BRIN), Indonesia, Letter Number: B-24/IPH.6/KS.02/IX/2020. The identification results stated that the type of rhizome used was white galangal (Alpinia galangal). According to Raina and Abraham (2017), galangal essential oil has been produced by the steam distillation method. The feeds were tested using in vitro from 5 treatments, namely elephant grass: concentrate (60:40) (control), 30, 60 and 120 無 galangal EO/300 mg dry matter feed and 5 無 pure cineol/300 mg dry matter feed. Cineol (purity 99%) was obtained from SIGMALDRICH. The commercial concentrate comes from the Jabung Agro Niaga Cooperative, Malang. The composition and nutritional content of feed ingredients are shown in Table 1. The Ethics Committee has approved all research procedures of the Faculty of Veterinary Medicine, Universitas Gadjah Mada, Letter Number: 0055/EC-FKH/Ex./2020.

Table 1. The composition and nutrient content of feed ingredients in the treatment

Nutrient content % (DM basis)

Mott elephant grass

Concentrate

Dry matter

18.79

91.65

Organic matter

76.21

88.42

Crude protein

13.57

14.38

Ether extract

2.77

2.60

Crude fiber

30.40

29.30

Nitrogen free extracta

29.82

42.03

Total digestible nutrientb

70.46

70.48

aNFE=Nett free extract, calculation results= 100-(ash+crude protein+ether extract+crude fiber) bTDN = total digestible nutrient; calculation results forage = 1.6899 + 1.3844(CP) + 0.7526(NFE) – 0.8279(EE) + 0.3673(CF), concentrates as an energy source = 2.6467 + 0.6964(CP) + 0.9194(NFE) + 1.2159(EE) – 0.1043(CF), source as a protein = -37.3039 + 1.3048(CP) + 1.3630(NFE) + 2.1302(EE) + 0.3618(CF)

In vitro digestibility and ruminal fermentation parameters

The rumen fluid for in vitro feeding was obtained from Bali cattle weighing about 300 kg. The feed given was in the form of fresh king grass as much as 25 kg/day and concentrate for beef cattle, 5 kg/day, which was given twice a day in the morning and evening. Feeding according to cattle needs for maintenance follows the National Research Council (NRC) standards. Rumen fluid was taken before morning feeding, then filtered and added to the media, according to Menke et al (1979). The syringe glass containing 30 ml of the fermentation medium. All glasses were incubated in a modified water bath at 39蚓 for 72 hours and the fermentation parameters were observed. At the end of 72 h incubation, the supernatant was centrifuged (10,000 g/10 min) to separate the microbial cells and the enzyme-containing supernatant. Measurement of amylase, CMC-ase was carried out according to Bergmeyer and Gawehn (1974) and protease using the method of Halliwell (1961).

In vitro digestibility analysis was measured using a 2-stage in vitro method (Tilley and Terry 1969). About 500 mg of the substrate was tested for crude protein and fiber digestibility, while 250 mg of the substrate was tested for the digestibility of dry matter and organic matter. The tube fermentation containing 10 ml of rumen fluid and 40 ml of McDougall’s solution (Tilley and Terry 1969), then incubated at 39蚓. At the end of the 48-h incubation, the fermented products were filtered and the residue was used to determine the nutrient digestibility of the rumen’s dry matter, organic matter, crude protein and crude fiber. After 48 hours of incubation, post-rumen digestibility was measured by adding three mL of 20% HCL and one mL of 5% pepsin, then incubated for 96 hours. Again, the fermented residue was analyzed for the digestibility of dry matter, organic matter, crude protein and crude fiber.

Statistical analysis

The data obtained were statistically analyzed using the R program software using a completely randomized directional pattern design. Differences were declared significant at p<0.05 (Steel et al 1997).


Results and discussion

Enzyme activities

The data in Table 2 shows that all doses of galangal essential oil addition significantly decreased p<0.05 on CMC-ase activity compared to controls. However, the CMC-ase activity significantly increased p<0.05 compared to the control at the cineol dose. Galangal essential oil supplementation at a dose of 120 無 significantly decreased p<0.05 the amylase enzyme activities significantly. The rumen’s fiber and starch digestion process involves CMC-ase and amylase produced by rumen bacteria, namely Ruminobacter amylophilus and Streptococcus Bovis (Wallace 2004). Doses of galangal essential oil addition decreased significantly by p<0.05 protease activity compared to controls. The activity of protease enzymes in the rumen is correlated with the abundance of proteolytic bacteria in degrading protein (Mahanani et al 2020). According to Kim et al (2017), proteolytic activity in the rumen is dominated by the Genus Prevotella. The decrease in amylase, cellulase and protease enzymes was due to galangal essential oil, but this did not occur in cineol. Galangal EO with cineole as the main compounds may relate to anti-microbes on bacteria and inactivation of enzyme activities in the rumen. Other components, such as phenol, may play a role in binding the enzyme’s active site. According to Young (2019), the hydroxyl group of phenolic compounds can bind to enzyme proteins. Furthermore, according to Zielińska-Błajet and Feder-Kubis (2020), each bioactive compound has different characteristics in binding proteins. Duval et al (2007) explained that essential oils with the main component of phenol have sensitivity to the bacteria Prevotella genus and R. Amylophilus, an amylolytic and proteolytic bacteria. According to Zhou et al (2020), thymol, a phenolic compound, is sensitive to Gram-negative bacteria in the rumen, namely Genus Prevotella. Another mechanism is that essential oils reduce bacterial colonization attached to the feed substrate. Wallace et al (2002) stated that the colonization of protease bacteria in the rumen could be inhibited by essential oils, which was indicated by a decrease in protease activity.

Table 2. Effect of galangal EO on enzyme activities by rumen in vitro

Measurements
(U/g)

Galangal EO

Cineole

(無)/300 mg (DM feed)

0

30

60

120

5

CMCase

5.90c0,32

4.51b0.17

3.09a0.30

3.07a0.23

11.55d1.03

Amilase

18.30c0,53

17.44bc0.75

16.90b0.30

13.03a0.48

18.35c1.07

Protease

39.49b0,84

38.80b1.32

32.14a1.59

29.84a1.84

43.18c0.61

In vitro nutrient digestibility

The addition of galangal essential oil to digestibility in vitro is presented in Table 3. Dry matter digestibility in the rumen decreased significantly by 28.49%, 34.58% and 32.66% at doses of 30, 60 and 120 無 galangal essential oil compared to controls (p<0.05). Meanwhile, there was no significant (p>0.05) compared to the control in the addition of cineole. The data also show a similar pattern for the digestibility of organic matter in the rumen. Crude protein digestibility in the rumen also decreased significantly by 21.87%, 26.13% and 45.75%. Furthermore, Crude fiber digestibility in the rumen decreased significantly by as much as 14.47%, 40.03% and 49.23% when galangal essential oil doses of 30, 60 and 120 無 were added. All the enzyme activities (CMC-ase, amylase and protease) in rumen are responsible for the digestion of fiber, starch and protein in the rumen. So that, the decreasing of their activities also effected on nutrient digestibility. According to Oskoueian et al (2013), essential oil supplementation in vitro fermentation decreased CMC-ase activity, followed by decreased fiber degradation. The essential oil in the ration could cover the feed substrate, especially fiber, so the enzymes could not make enzyme-substrate complexes (Duval et al 2007).

The decreasing nutrient degradability in rumen that occurs due to the addition of galangal essential oil is possible to decrease the abundance of proteolytic bacteria from the Prevotella genus. This genus is associated with the fermentation of xylan hemicellulose, pectin and protein and peptide metabolism. Thus, decreased CMC-ase and protease enzyme activity decreased protein and fiber digestibility. According to Bach et al (2005), the attachment of proteolytic bacteria is the beginning of the degradation of feed protein. Research using eucalyptus essential oil at a dose of 40 無 equivalent to cineol 12.17% reduced the digestibility of organic matter by 14.52% (Chouchen et al 2018). However, according to Abdelrahman et al (2019), adding eucalyptus essential oil at a dose of 40 無 equivalent to 35.82% cineol did not affect the digestibility of organic matter in rumen fermentation in vitro. The difference in cineol levels between the two studies was due to the different species of eucalyptus oil. Similar to this study, the digestibility of organic matter also decreased with the addition of galangal essential oil with a cineol content of 24%. Therefore, it can be concluded that in both types of essential oils with sources of cineol that are not too high, other compounds may play a role other than cineol in rumen microbial modification.

Table 3. Effect of galangal EO on nutrient digestibility by rumen in vitro

Measurements

Galangal EO

Cineole

(無)/300 mg (DM feed)

0

30

60

120

5

Dry matter digistibility (%)

Rumen

47.91c0.26

34.26b0.53

31.34a0.11

32.26ab0.56

48.51c1.31

Post

10.32a0.88

18.58b1.04

17.92b0.94

10.58a0.35

13.09a1.78

Total

58.23d0.85

52.84c0.75

49.26b0.83

42.84a0.72

61.61e1.13

Organic matter digestibility (%)

Rumen

52.55d0.22

41.13c0.32

33.63b0.13

29.59a0.20

53.47d1.08

Post

3.56a0.30

7.80bc0.92

10.45c1.41

8.89bc0.16

5.69ab0.49

Total

56.11d0.62

48.94c0.71

44.09b1.40

38.48a0.16

59.16e0.43

Crude protein digestibility (%)

Rumen

62.68c1.41

48.97b0.76

46.30b1.82

34.01a0.82

63.24c0.21

Post

15.09b1.05

18.86bc0.36

17.53c0.90

34.83d1.60

12.86a0.42

Total

77.78c 0.48

67.83b1.08

63.83a1.17

68.84b1.28

76.10c0.26

Crude fibre digestibility (%)

Rumen

48.36d0.68

41.36c0.82

29.00b0.44

24.55a0.81

54.61e0.67

Post

4.57b0.81

6.65c0.87

17.17d1.78

18.05d0.28

2.63a0.92

Total

52.94d0.30

48.01c0.24

46.17b1.77

41.05a0.64

57.25e0.81

In post-rumen, dry matter digestibility increased significantly by 44.45% and 42.41% at 30 and 60 無 galangal essential oil doses, respectively (p<0.05). Meanwhile, the addition of 120 無 of galangal essential oil and cineol had no significant p>0.05 compared to the control. Furthermore, protein digestibility increased significantly by p<0.05 at 19.98%, 13.91%, 56.67% at 30.60 and 120 無 of galangal essential oil. Furthermore, the addition of cineol caused the post-rumen protein digestibility to decrease significantly by 14.77% compared to the control (p<0.05). Meanwhile, with the addition of pure cineol, fiber digestibility in post-rumen was increased by 11.44% compared to the control. Differences in fermentation patterns between galangal essential oil and cineol can be caused by differences in the mechanism of action on the abundance of cellulolytic bacteria or cellulose enzyme activity. According to Machado et al (2014), the components of pure monoterpenes compounds have lower antibacterial properties than essential oils. It was further explained that essential oil compounds, both major and minor components, work synergistically as antibacterial. Several studies using essential oils also reported the same results: a decrease in fiber digestibility (Gnal et al 2017; Kurniawati et al 2020). The decrease in fiber degradation in the rumen is possible due to the inactivation of cellulolytic enzymes by bioactive compounds from galangal essential oil. It was explained by Benchaar et al (2008) that essential oils can bind substrates and feed particles. Methane production is positively correlated with fiber digestion (Deramus et al 2003), so it is a possibility that galangal EO declined methane production.


Conclusions


Acknowledgments

The research works are supported by the Doctoral Scholarship from the Ministry of Agriculture, Indonesia.


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