Enhanced conjugated linoleic acid and biogas production after ruminal fermentation with Piper betle L. supplementation

Piper betle L. is edible plant richer in polyphenols that might improve feed utilization in rumen diet. The objective of the present study was to investigate the effect of various Piper betle L. powder (PL) doses on in vitro rumen microorganisms, ruminal biogas and fermentation end-product production, and biohydrogenation including lipolysis-isomerization. The completely randomized design used five levels of PL supplementation (0, 25, 50, 75 and 100 mg DM) incubated with 400 mg of a basal substrate of Pangola hay and concentrate (50:50). The matrix compounds (g/kg DM) of 0.27 catechin, 0.11 rutin, 3.48 quercetin, 0.41 apigenin, 0.04 myricetin, 0.27 kaempferol, 0.76 eugenol and 0.22 caryophyllene derived from PL altered the fermentation pattern, with an increase in degradable nutrients and total volatile fatty acids and acetogenesis without shifting pH during fermentation. These values promoted in vitro gas production, with higher carbon dioxide and lower methane production. Although, hydrogen recovery from lipolysis-isomerization in biohydrogenation was limited, PL successfully promoted stearic acid (C18:0) accumulation by changing the biohydrogenation pathway of fatty acids, causing more C18:1 trans-11 rather than C18:2 trans-11, cis-15. Consequently, this resulted in more conjugated linoleic acid (CLA) cis-9, trans-11, CLA trans-10, cis-12 and CLA trans-11, cis-13. Enhanced PL supply increased total bacteria and fungal zoospores due to a reduction in rumen protozoa. In conclusion, our results demonstrated that PL is a feed additive with potential for ruminants, promising improved ruminal fermentation and biohydrogenation, while reducing methane production.


INTRODUCTION
Piper betle L. is tropical, edible and affordable plant which is reported to contain a host of potent polyphenol compounds, such as flavonoids Purba et al. reported that Australian plants selected as CLAdegrading inhibitors were successful in targeting ruminal bacteria such that biohydrogenation yielded more CLA. Unfortunately, the non-specific bioactive compounds were not reported. Since Piper betle L. has other flavonoids and essential oils (namely catechin, rutin, apigenin, myricetin, kaempferol and caryophyllene), it was hypothesized that Piper betle L. might potentially change fermentation pathways with regard to pyruvate stage and biohydrogenation. Therefore, the objective of present study was to investigate the influence of various inclusions of Piper betle L. powder (pL) on in vitro rumen microorganisms, ruminal biogas and fermentation end-product production, as well as biohydrogenation including lipolysis-isomerization.

Animal, feed, Piper betle L. powder and experimental design
Piper betle L. leaves were planted, harvested and sampled from random spots at SUT Organic Farm, Nakhon Ratchasima, Thailand (14°52'48''N, 102°00'54''E at an elevation of 243 m above sea level). Leaves were removed from the plant and kept at 4 °C overnight to avoid any nutrient destruction (PURBA & PAENGKOUM, 2019). The leaves were mechanically ground (Retsch SM 100 mill; Retsch Gmbh, Haan, Germany) into a powder (pL) and particles were passed a 1-mm sieve, and placed into plastic, sealed pouches in the desiccator (Auto dry desiccator model OH-3S, SPC RT, Thailand) until usage time. In addition, three female Saanen goats (40 ± 1.51 kg body weight) were prepared as rumen donors, with ad libitum access to water and a maintenance total mixed ration (TMR) consisting of Pangola hay and concentrate (50:50) (NRC, 2007). This feed was also presented as a basal substrate in the in vitro experiment. Animals were allocated to the TMR for 20 d, including a 15 d adaptation period and 5 d of sampling. Formulation and chemical composition of the PL and feeds (basal substrate) are presented in table 1. The study was designed as a completely randomized design with five levels of pL at 0, 25, 50, 75 and 100 mg DM, incubated with 400 mg of the basal substrate of TMR and hay (50:50).

In vitro experiment and sampling
The in vitro experiment was conducted using the Hohenheim gas test method of MENKE & STEINGASS (1988), as modified by pAENGKOUM et al. (2015). On the 16 th , 18 th and 20 th day of the feeding trials, rumen fluid was extracted from the rumen via oral lavage by suction pump (Hitachi CV-SF18, Japan) before morning feeding time, avoiding saliva collection (LODGE-IVEY et al., 2009). Rumen fluid was strained using a nylon membrane (400 µm; Fisher Scientific S.L., Madrid, Spain) into Conical flask, and mixed with salivary buffer (1:2, ml:ml) under CO 2 and kept at 39 °C for 24 h. The composition of the rumen fluid buffer mixture was as follows: 474 ml rumen fluid, 0.60 g MgSO 4 .7H 2 O, 1.32 g CaCl 2 .2H 2 O, 0.10 g MnCl 2 .4H 2 O, 0.10 g CoCl 2 .6H 2 O, 0.80 g FeCl 3 .6H 2 O, 35 g NaHCO 3 , 4 g NH 4 HCO 3 , 5.70 g Na 2 HPO 4 , 6.20 g KH 2 PO 4 , 10 mg resazurin and 0.40 g NaOH, made up to 1000 ml with distilled water (MENKE & STEINGASS, 1988). The incubation was prepared using a 100 ml glass syringe filled with basal substrate, the pL dose and the rumen fluid/buffer mixture. For example, the treatment group using 100 mg of PL contained 400 mg substrate plus 100 mg pL, mixed with 30 ml rumen fluid/buffer. The sample for incubation was complete after 8 g/kg DM sunflower oil was infused into each glass syringe. Ten replicates of each treatment were prepared in three consecutive runs, with three blank controls added per run, containing only the rumen fluid/buffer mixture.
After 24 h of incubation, total gas production was read based on the model of ORSKOV & MCDONALD (1970). A 30 ml sample of gas from the glass syringe was used to measure methane and carbon dioxide levels by gas chromatography (Agilent 7890A, USA). The glass syringe was kept on ice to impede fermentation (JAYANEGARA et al., 2012a). Once the glass syringe was uncapped, the pH of the fermented content was measured directly using a pH meter (Oakton 700, USA). The fermented content was divided into four portions. The first portion was centrifuged at 6,000 × g at 4 °C for 15 min and the supernatant was stored at -20 °C. For volatile fatty acid (VFA) analysis, the supernatant was fixed with 25% metaphosphoric acid (ERWIN et al., 1961;FILÍpEK & DVOŘÁK, 2009). Rumen ammonia detection was performed in accordance with the micro-Kjeldahl method (Foss Kjeltech 8100, USA) (AOAC, 2005). The second portion was stored at -20 °C for fatty acid (FA) analysis. The third portion was treated with 10% formalin solution in a sterilized 0.9% saline solution for observation and calculation of total bacteria, protozoa and fungal zoospores using a counting chamber (Neubauer, Germany) as described by WANApAT et al. (2013). The last portion was prepared for in vitro dry matter degradability (IVDMD) and in vitro organic matter degradability (IVOMD) following the method of TILLEY & TERRY (1963) using pre-weighed Gooch crucibles (Isolab, Gmbh, Germany).

Laboratory analysis and calculation
The samples of substrate and pL were chemically analyzed for dry matter (DM), organic matter, ash, crude protein (total N × 6.25) and crude fat following prior protocols (AOAC, 2005). Acid detergent fiber and neutral detergent fiber were measured as described previously (VAN SOEST et al., 1991), with residual ash included. The gross energy was determined using a bomb calorimeter with an O 2 carrier (Parr 6200 bomb calorimeter, Parr Instruments Co., Moline, IL) according to the manufacturer's instructions. Concentrations of rutin, apigenin, quercetin, kaempferol, myricetin, eugenol and caryophyllene was determined by a mean peak rate of signal following the High performance Liquid Chromatography with Diode Array Detector principle (PURBA & PAENGKOUM, 2019). All measurements were performed in triplicate and chemical standards were included in each analytical run as appropriate (Table 1).
Fatty acid (FA) measurement was conducted following the guidelines of FOLCH et al. (1957), as modified by (DE WEIRDT et al., 2013) using gas chromatography (Agilent 7890A, USA) with a Cp-Sil88 column for FA methyl esters (100 m × 0.25 mm × 0.2 µm; Chrompack Inc., Middelburg, the Netherlands). Heptadecanoic acid (C17:0; Sigma-Aldrich) was applied as an internal standard (LASHKARI & JENSEN, 2017) and the C17:0 value was excluded in the calculation. The column temperature was kept at 70 °C for 4 min, then increased by 13 °C/min to 175 °C and held for 27 min, then increased by 4 °C/min to 215 °C and held for 17 min, then increased by 4 °C/min to 240 °C and held for 10 min. More detailed information about the detection and calculation methods of peak area is given by LOURENÇO et al. (2014). The FA profile of feed and PL is presented in table 1. Calculation of the efficiency of lipolysis and isomerization (C18:2 n-6 → C18:2 cis-9, trans-11), and the hydrogenation processes of C18:2 n-6 and C18:3 n-3 in incubations were calculated as determined by BOECKAERT et al. (2007), as modified by pANYAKAEW et al.  Table 4).

Statistical analysis
Statistical analysis accounted for the completely randomized design using the pROC GLM procedure of SAS 9.4. Data were analyzed using the model: where Y ST = the dependent variable, A = the overall mean, B S = the influence of the various pL doses ( S =1-5), and E ST = the residual effect. Because the runs were not significantly different, results are presented as mean values with the standard error of the mean. Differences between treatment means were calculated by Tukey's test (KAPS & LAMBERSON, 2004). Orthogonal polynomial contrasts were used to estimate the linear and quadratic pL effects. All differences with p<0.05 were accepted as representing statistically significant differences.

Ruminal biogases, fermentation end-products and microorganisms
The effect of the inclusion of Piper betle L. powder (pL) on fermented substrate in terms of ruminal biogas, fermentation end-products and microorganisms after 24 h, is presented in table 2. The present matrix of flavonoids and essential oils derived from PL gradually increased total volatile fatty acids (VFAs) corresponding to an increase in degradability, especially of fermented organic matter. In this study, in vitro dry matter degradability (IVDMD) and in vitro organic matter degradability (IVOMD) rose with the increase of pL. These results increased the total VFAs. However, unchanged numbers of total VFAs and degradability efficiencies were found previously, where they were compared to the control after substrate was incubated with quercetin, rutin and eugenol (CASTILLEJOS et al., 2006;OSKOUEIAN et al., 2013). Contradictions could be due to different basal substrates in proportion to the bioactive plant compound. The former studies subjected the ruminants Purba et al. to a 60:40 forage: concentrate diet (CASTILLEJOS et al., 2006;OSKOUEIAN et al., 2013), whereas in our study, a 50:50 Pangola hay: concentrate diet was applied. Also, those reports purchased a commercial secondary compound product. Notably, in vitro studies with supplementing active plant compound to modulate rumen performance may result in varied outcomes due to different purity rate, binding behavior and hydroxyl compound (pURBA et al., 2020). Despite pL derived from conventional procedure, pL had been reported owning abundant hydroxyl groups and as carbohydrate sources that enhanced the fermented substrate during fermentation (PURBA & PAENGKOUM 2019). Hence, this elaboration may also account for why the final results were slightly different. Higher VFAs values reflect a greater amount of fermented substrate by rumen microbes that could also be recognized in the total production of gas during fermentation. In this study, gas production was promoted by the addition of pL, with more carbon dioxide and less methane gas being produced. A reduction in methane production due to failed methanogenesis through the use of flavonoids and essential oils has been reported in an earlier review (pATRA & SAXENA, 2010), but the role of rutin, apigenin, myricetin, kaempferol and caryophyllene was not discussed. A smaller proportion of methane gas seems to imply that rumen methanogenesis occurred sluggishly in this study, causing a shift of the profile of VFAs, particularly the acetate, propionate and butyrate fractions.
In the present study, PL during fermentation resulted in higher acetate and butyrate production, but lower propionate, valerate and branched-chain VFAs (the iso-acid fraction). As calculated, this study also showed an inhibited hydrogen supply (HR) to methanogenesis and biohydrogenation. According to prior studies, propionate produced from pyruvate increases hydrogen consumption, and results in less efficient methanogenesis (MOSS et al., 2000;TAVENDALE et al., 2005). However, a meta-analysis by JAYANEGARA et al. (2012b) reported no significant relationship between the propionate surge and methane inhibition. GREENING et al. (2019) confirmed that hydrogen metabolism is a more complex and widespread trait among rumen microorganisms, and claims that the hydrogen yield is usurped by other consumers (not only methanogens) in fumarate and nitrite reduction (Selenomonas spp.) and acetogenesis (Blautia spp.). In the present study, PL supplementation increased the acetate fraction, providing more evidence that hydrogen was consumed by acetogenesis (Blautia spp.) and hydrogen supply was depleted for this reason. Valeric and iso-acid fractions in a recent study (ANDRIES et al., 1987) were lower as consequence of less propionic acid. All changes in fermentation behavior above occurred with an unchanged ruminal pH across all treatments, while an alteration in pH  Linear Quadratic pH 6.9 6.8 6.8 6.9 6.9 0.013 0.438 1.000 Total gas production (ml/g OM) 30.  Table 2). The range of pH and ammonia concentration in the present study was 6.8-7.0 mg/100 ml and 19.9-20.1 mg/100 ml, which was acceptable for ruminal microorganisms to survive by modulating microbial growth and fermentation efficiency (WANApAT & pIMpA 1999). Enhanced pL supply reduced protozoa in the rumen (Table 2). A decrease in protozoa number due to the supplementation of secondary compounds has been reported before (CASTILLEJOS et al., 2006;ZHOU et al., 2011;OSKOUEIAN et al., 2013;ANDRÉS et al., 2016;SZCZECHOWIAK et al., 2016) and a primary reason is the ability of bioactive compounds to alter cell wall synthesis or nucleic acid synthesis of protozoa. This reduction in protozoa may be reason why the bacteria and fungal zoospores increased within the incubation period. DEHORITY (2003) mentioned protozoa engulfing rumen bacteria at approximately 20,000 cells per hour. Thus, increased bacteria numbers were contingent on the eliminated protozoa. NEWBOLD et al. (2015) also reported that fungal zoospores were inversely proportional to protozoal number, indicating a competitiveness between protozoa and fungal zoospores for substrate during fermentation. In another report, CHERDTHONG et al. (2019) confirmed that fungal zoospores had a defensive mechanism for survival in the presence of bioactive compounds.
Clearly, the present matrix of flavonoids and essential oils derived from PL inhibited biohydrogenation, where the biohydrogenation of C18:2n-6 and C18:3n-3 was abated, as poly unsaturated fatty acids (pUFAs) were observed. Reference to this shift in biohydrogenation, including the isomerization of FA by lipolysis, using flavonoids and essential oils of pL, is relatively limited. The application of only phenolic acid, quercetin, anthocyanin and eugenol for in vitro study is available in the literature (LOURENÇO et al., 2014;YANZA et al., 2018;BRYSZAK et al., 2019). In our study, PL encouraged rumen biohydrogenation to yield a more considerable PUFA accumulation, e.g. CLA cis-9, trans-11, CLA trans-10, cis-12 and CLA trans-11, cis-13, less saturated fatty acid as C18:0 and less mono-unsaturated fatty acid accumulation of C18:1 trans-11, rather than C18:2 trans-11, cis-15. It is reported that the biohydrogenation pathway could be shifted by the provision of flavonoids in the animal diet (YANZA et al., 2018), resulting in an increased amount of CLA cis-9, trans-11 produced in rumen fluid and milk (BRYSZAK et al., 2019). As shown in table 4, enhanced pL supply interacted in the first biohydrogenation pathway of C18:2n-6, resulted in the accumulation of C18:2 cis-9 rather than C18:2 trans-11, cis-15, which was observed to be different to other studies (LOURENÇO et al., 2014). The latter study reported that quercetin and eugenol were unable to alter the proportion of C18:2 cis-9 through the first biohydrogenation pathway of C18:2n-6, although, it seemed successful at a slight reduction in C18:2 trans-11, cis-15. However, later biohydrogenation regarding the transformation of C18:1 trans-11 to C18:0 in these reports showed similar outcomes, addressing the limited supply of C18:1 trans-11, leading to a decrease in stearic acid (C18:0) accumulation. A possible reason for this difference concerns the FA isomer in the diet. LOURENÇO et al. (2014) fed rumen fermenters with more C18:3n-3 than C18:2n-6. In contrast, the present study provided an abundance of C18:2n-6 from sunflower oil. This assertion could be true if the FA component was a major factor in modulating biohydrogenation (ROY et al., 2013). However, biohydrogenation efficiency is more complex because this process promotes rumen biohydrogenation bacteria and should be wisely considered for other factors, such as hydrogen supply (LOURENÇO et al., 2010). In this study, stearic bacteria (that were encouraged by the presence of pL) were responsible for undertaking CLA formation, but identification of specific ruminal microorganisms to confirm this was outside the scope of the present study. It is possible that small protozoa and big bacterial populations could influence lipase activity (LOURENÇO et al., 2010). Therefore, the change in rumen microorganisms, especially bacteria and protozoa, should not be ignored.

CONCLUSION
This study demonstrated that the matrix of flavonoids and essential oils in Piper betle powder can stimulate conjugated linoleic acid accumulation from biohydrogenation products, without interrupting nutrient fermentation. Methane production is also reduced. Another perspective of the use of these compounds can be assessed in vivo, where animals are fed with these bioactive compounds. It might be an alternative strategy to improve the quality of ruminant feeds. Table 4 -The mean value of substrate incubated with Piper betle L. powder (pL) on the efficiency of lipolysis + isomerization (C18:2 n-6 → C18:2 c9t11) and hydrogenation processes of C18:2 n-6 and C18:3 n-3 in incubations.