Garlic as a rumen modifier for eco-friendly and economic livestock production

Abstract

Allium sativum (garlic) has been very popular in Asian kitchen as one of the major spices. The strong smelling juice of bulb contains volatile oils composed of sulphur-containing compounds: allicin, diallyl disulfide and diallyl trisulfide, which are responsible for anti-microbial activity. Recently garlic, its oil, extracts in different organic and aqueous solvents and individual components of essential oils have been examined as a feed additive to manipulate rumen function for controlling enteric methane emission and improving feed conversion efficiency. The results reported so far indicate that garlic and its essential oils inhibit methanogenesis significantly accompanied with a lower acetate to propionate ratio indicating a diversion of fermentation in a favourable direction. As methanogenesis is the major hydrogen sink in the rumen, its inhibition requires the disposal of reducing equivalents produced during fermentation of feed. The propionate synthesis serves as an alternate hydrogen sink. The anti-microbial compounds present in garlic appear to be selective inhibitors of methanogenesis, as there is no adverse effect on feed degradation in the rumen. Garlic and its oil adversely affect the protein degrading bacteria and deamination activity of the rumen contents. Only a few in vivo experiments have been conducted using garlic as a feed additive, and it appears to have good potential for rumen manipulation for eco-friendly (with minimum methane emission) and economic livestock production.

Keywords:

• garlic
• Allium sativum
• methane
• rumen
• feed additive
• plant secondary metabolites

Introduction

The origin of garlic (Allium sativum) is reported to be as old as 5000 years when it was cultivated in Egypt and Mediterranean region. In India and China its use in kitchen and Ayurvedic and Chinese medicines started by 600 B.C. The Spanish, Portuguese and French introduced it to the rest of the world. Presently it is grown in temperate and tropical regions all over the world and many different cultivated types and strains have been developed to suit different climates. China is the largest producer of garlic (11.0 million tons annually) accounting for more than 75% of total production in the world. India is the second largest producer of garlic but far behind China with annual production of only 0.5 million tons (FAO 2005). Korea, USA, Russia, Egypt, Spain, Argentina and Ukraine are the other countries which produce good amounts of garlic for local consumption and exports. Allium sativum belongs to the order Asparagales and the family Alliiaceae. All plant parts, except root attached to bulb and the protective thin layer over the surface, are edible when young and tender.

Chemical composition

Garlic contains a complex mixture of many secondary metabolites which include allicin (C₆H₁₀S₂O), diallyl sulfide (C₆H₁₀S), diallyl disulfide (C₆H₁₀S₂) and allyl mercaptan (C₃H₆S) (Lawson 1996). Along with volatile oils (0.1–0.36%), it also has a good amount of protein (16–17%). In addition to that it is a rich source of sulphur, potassium, phosphorus, magnesium, sodium and calcium. The sulphur content in garlic varies from 0.5 to 3.7 g per 100 g of dry matter. Garlic cloves are rich in alliin, a cysteine-based sulphur-rich amino acid present abundantly in the mesophyl tissues. The enzyme which degrades alliin to allicin, known as alliinase is also present in the garlic cloves, but it is concentrated in vascular bundle sheath and is, therefore, kept away from the substrate (alliin) by the cellular walls between the two tissue types. Therefore the intact garlic cloves smell only mildly, but the characteristic garlic smell intensifies on crushing. The slicing, mincing or crushing brings the substrate (alliin) in contact with the enzyme (alliinase) and the product allicin is formed, which is primarily responsible for the characteristic garlic smell. But allicin is an intermediate volatile molecule, which decomposes to other compounds like diallyl sulfide, diallyl disulfide and diallyl trisulfide, dithiins and ajoene (Amagase et al. 2001). This instability of allicin is responsible for low level of allicin (<1%) in garlic bulb (Lawson 1996). Allicin remains active in the crushed garlic only for a few hours, due to its utilisation in further biochemical reactions and also because of its volatile nature. The characteristic smell and anti-microbial activity also decreases as the concentration of allicin in the mixture decreases. The polysulphides which are among the last breakdown products of allicin have the least taste, smell and the least immediate anti-microbial effects. Cooking also destroys anti-microbial characteristics of allicin. A wide range of different organosulphur compounds are produced during processing of garlic including thiosulphinates, volatile organosulphurs and water-soluble organosulphurs (Kodera et al. 2002). Garlic products can be classified into four groups: garlic essential oils, garlic oil macerate, garlic powder and garlic extract (Amagase 2006).

Anti-microbial activity

Garlic has several medicinal properties, but its anti-microbial activity against different pathogenic microbes is perhaps the most widely studied scientifically, using modern techniques of microbiology. The earliest report on anti-microbial effect of garlic is the studies of Louis Pasteur in 1858, who examined its anti-bacterial effect in in vitro conditions. Garlic acts as only a mild antibiotic in comparison to modern antibiotics, but added advantage of its use as antibiotic is that the microorganisms do not develop a resistance against garlic as they do against the conventional antibiotics. The antibiotic properties of garlic are a direct result of allicin produced from raw and crushed garlic, which is destroyed by cooking or boiling (Sato and Mitaya 2000). The cooked garlic has no antibiotic value although it still retains other benefits.

It is a natural anti-microbial agent effective against several bacteria (Bacillus, Escherichia, Mycobacterium, Pseudomonas, Staphylococcus and Streptococcus), fungi, yeasts, small insects and parasites. In vitro screening experiments have shown sensitivity of some important bacteria like Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae to the presence of fresh garlic extracts (Farbman et al. 1993; Ankri and Mirelman 1999). The main anti-microbial effect of allicin is due to its oxidative interaction with important thiol-containing enzymes (Cavallito and Bailey 1994; Pérez-Giralo et al. 2003).

The anti-microbial activity of phenolics of garlic extracts against bacteria (Staphylococcus aureus, Salmonella enteritidis) and fungi (Aspergillus niger, Penicillium cyclopium and Fusarium oxysporum) was demonstrated to be more potent than onion and therefore recommended to be used as a food preservative (Noureddine et al. 2005). Ross et al. (2001) compared anti-microbial activity of garlic oil and garlic powder against human pathogens and found that garlic powder thiosulphinates have more potential than garlic oil sulphides, but to combat human gut infection garlic oil is more effective because of its higher affinity to cysteine.

Garlic as anti-cholesterol agent

A demonstration on the adverse effect of garlic oil on the activity of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase enzyme leads to the anti-cholesterol activity of garlic, because the enzyme is responsible for cholesterol production in human beings. It has been demonstrated that by inhibiting the activity of this enzyme by including antibiotics like lovastatin and mevastatin, cholesterol production can be controlled (Miller and Wolin 2001). Anti-cholesterol activity of garlic was reported by Eilat et al. (1995) in rabbits and by Qureshi et al. (1983) and Konjufca et al. (1997) in chicken. Seo et al. (2009) demonstrated improvement in insulin resistance, decrease in serum total cholesterol and triglyceride and increase in HDL-cholesterol levels in db/db mice (an animal model of type 2 diabetes) fed on diet supplemented with 5% aged black garlic powder, a garlic product which is prepared by aging whole garlic at high temperature (70^°C) and humidity (90% RH) (Jang et al. 2008). Lee et al. (2009) also suggested that aged black garlic can be useful in ameliorating the diabetic complications.

Garlic as a prebiotic

In addition to broad spectrum anti-microbial activity, garlic also acts as a prebiotic, a nutrient which is not digested in the gut but acts as a stimulator for the beneficial gut microbes leading to improved microbial eco-system. In addition to volatile essential oils, Allium sativum is a rich source of inulin, a storage oligomer of fructose and a very potent prebiotic. Inulin is not degraded in gastrointestinal tract (GIT) and is therefore available as substrate for the microflora of large intestine. Several reports have demonstrated the stimulatory effect of inulin on Biofidobacterium and maintaining low levels of pathogen (E. coli, Clostridium) population (Wang and Gibson 1993). Feeding of inulin resulted in a higher production of hydrogen without methane emission but accompanied with a four-fold increase in butyrate concentration in comparison to fibrous diet (Roland et al. 1995). The inulin present in garlic is highly active. The inulinase activity of Streptococcus spp. was tested using different sources of inulin including a pure product, but highest activity was found with garlic powder as a source of inulin (Sharma et al. 2006).

Garlic as anti-methanogenic and anti-protozoal agent

Methanogenesis is an essential metabolic process in the rumen to remove hydrogen produced during fermentation of polysaccharides resulting in production of methane which accounts for 2–12% loss of dietary energy (Johnson and Johnson 1995). Methane is also one of the potent greenhouse gases contributing significantly to the environmental pollution. In the total global methane efflux, the livestock contributes about 20% (gut microbial and animal waste fermentation) and the Indian livestock's contribution is about 11% of total contribution of livestock in the world.

Patra et al. (2006) evaluated ethanol, methanol and water extracts of garlic bulb in in vitro gas production test using rumen liquor of buffalo as inoculum and reported 55% inhibition in methane emission with the methanol extract (0.5 ml/30 ml incubation medium) without affecting protozoa population (Figure 1). Since the majority of methanogens are closely associated with protozoa (Finlay et al. 1994), methane inhibition without affecting protozoa means the inhibition was due to direct attack on methanogens, which are free living in the liquid portion of rumen content. The four major components of garlic were tested individually for their effect on in vitro methanogenesis and was found that only garlic oil, diallyl disulfide and allyl mercaptan were able to inhibit methane emission, whereas allicin was ineffective (Busquet et al. 2005a, b, 2006; Kamel and Greathead 2008). They suggested that the organosulphur compounds of garlic oil could inhibit rumen methanogenic archaea by inhibiting the enzyme HMG-CoA reductase. The archaea including methanogens have unique membrane lipids formed of long chain isoprenoid alcohols and the formation of isoprenoid units is catalysed by HMG-CoA enzyme. The results indicated that methane emission was adversely affected without disturbing rumen fermentation with inclusion of allicin in the reaction mixture, which confirms earlier reports of Patra et al. (2006) who used garlic extracts in in vitro gas production test and reported significant reduction in methane emission without affecting dry matter degradability. Gracía-González et al. (2007) screened a number of plants, herbs and spices (148 samples) for their anti-methanognic activity in vitro. Most of the plants were ineffective but inclusion of garlic at the rate of 135 mg/g of substrate resulted in more than 20% inhibition in methane production, with no affect on gas production and slight increase (2%) in in vitro dry matter (DM) degradability. Kamel et al. (2009) have demonstrated that the effect of inclusion of garlic oil on in vitro methane and volatile fatty acids (VFA) production is diet and dose dependent.

Figure 1.  Effect of methanol extract of garlic on in vitro gas and methane production and dry matter degradability.

Feeding of garlic bulb at the rate of 1% of dry matter intake resulted in 11% inhibition in methane emission (l/kg DDMI) in sheep (fed on concentrate roughage ratio of 1:1) as estimated in an open circuit respiration chamber (Patra et al. 2011) along with an increase in nutrient digestibility. The activity of the plant secondary metabolites is also associated with pH, type of diet of the animal; therefore, the dose of the feed additive has to be standardised with the specific feeding regimen of the animal (Calsamiglia et al. 2007).

Ferme et al. (2007) reported changes in methanogen community by garlic extracts. They identified garlic oil (particularly diallyl disulfide) as first plant extract which selectively acted upon methanogens. A recent report by Ohene-Adjei et al. (2008) indicated that inclusion of essential oils (cinnamaldehyde, garlic oil, juniper berry oil) in the diet of Canadian Arcott ewes, fed on barley-based diet, did not affect methanogen population as evidenced by total copy number of archaeal 16S rRNA thus not affecting methanogenesis, but there was an increased diversity of methanogenic archaea related to Methanosphaera stadtmanae, Methanobrevibacter smithii and some uncultured groups. The authors attributed this change in methanogen diversity to the changes in protozoal population. Recently a study was conducted using rumen simulation technique to explore the anti-methanogenic activity of commercially available allicin. At 20 µl/ml level of allicin, the total volatile fatty acids (TVFA) and ammonia nitrogen were not affected but methane production was reduced. Real-time PCR analysis revealed reduction in methanogen DNA indicating reduced methanogen numbers but total bacteria were not affected (McAllister and Newbold 2008). The response of animals to the feeding of essential oils might be dose dependent.

Effect of garlic on rumen fermentation

The feed is fermented in the rumen to produce volatile fatty acids, microbial protein, carbon dioxide, methane and other by-products. The volatile fatty acid pattern reflects the efficiency of nutrient utilisation, as among the three volatile fatty acids, acetate and butyrate synthesising pathways are hydrogen producers and propionate pathway is hydrogen consumer and therefore an increase in propionate production is generally associated with a decrease in methane emission, thus increasing the efficiency of feed utilisation by the animals (Russell 1998).

The inclusion of ethanol and methanol extracts of garlic in in vitro incubation with buffalo rumen liquor shifted the volatile fatty acids production towards more propionate, resulting in a decreased acetate to propionate ratio (Patra et al. 2006, 2009) (Figure 2). Feeding of garlic bulb at the rate of 1% of dry matter intake to buffalo did not affect volatile fatty acids production (Patra et al. 2007), which might be due to the high level of secondary metabolites in in vitro experiment and the level of feeding (1% of feed intake) might not be sufficient to induce similar effects on in vivo methane emission and fermentation of feed in the rumen. Busquet et al. (2005a) reported a decreased proportion of acetate and branched chain fatty acids and increased proportion of propionate, butyrate, small peptides and amino acid nitrogen by inclusion of garlic oil (300 mg/l) in continuous culture system. To elucidate the mode of action of various components of the garlic oil, Busquet et al. (2005b) tested graded levels of garlic oil, diallyl sulfide, diallyl disulfide, allicin, allyl mercaptan in three different systems, that is, in vitro batch fermentation, in vitro continuous culture and in vitro gas production. In all the three experiments, decreased acetate with increased butyrate was a common observation with inclusion of any of the fractions of garlic but propionate production was variable as with garlic oil and diallyl disulfide there was an increased propionate production, whereas with allyl mercaptan there was no affect on propionate. Busquet et al. (2006) reported no effect on branched chain VFA, total VFA but reported an increased propionate and butyrate production and decreased acetate production by adding garlic oil at the rate of 300 mg/l in in vitro batch culture. The authors were of the opinion that decreased acetate to propionate ratio gave indication that this compound can inhibit methane production either by directly inhibiting methanogenic archaea or by inhibiting methanogenesis. The results indicated that the effect of garlic was more pronounced as compared to its active components individually. In all the experiments the highest dose (3000 mg/l) of garlic and its active components was detrimental which might be due to its antimicrobial activity. Gracía-González et al. (2007) reported that the inclusion of garlic bulb in the substrate in in vitro test using serum bottles did not affect TVFA, but acetate to propionate ratio decreased significantly. The dry matter disappearance increased significantly, whereas neutral detergent disappearance remained unchanged. The inclusion of garlic and other plant extracts like cinnamon, oregano and anise affected total VFA and individual acids between day 2 and 6 of fermentation in a continuous-culture fermenter using rumen liquor from dairy cows as inoculum. After 6 days these changes in VFA pattern diminished and was considered as adaptation period of microbes to these additives. With garlic extract there was an accumulation of amino acid nitrogen and reduction in ammonia nitrogen, which was attributed to inhibition of deamination. The authors suggested that the short-term in vitro studies with the natural plant products must be misleading (Cardozo et al. 2004). Busquet et al. (2006) and Castillejos et al. (2007) were also of the opinion that short-term batch culture studies were not sufficient for the biological expression of essential oils as it has been observed that in long-term in vitro trials the rumen bacteria might get adapted to these compounds. Therefore the potential use of the plant extracts can be elucidated only by long-term in vivo feeding trials.

Figure 2.  Effect of methanol extract of garlic on volatile fatty acids and protozoa.

Feeding of graded levels of garlic powder (80–120 g/day) along with urea treated rice straw to ruminants resulted in a decreased acetate:propionate ratio but total volatile fatty acids, ammonia nitrogen and pH of rumen liquor were not affected. However, nitrogen balance was significantly increased in a dose-dependent manner. Garlic powder also exhibited anti-protozoal activity since protozoa population decreased in the supplemented group (Wanapat et al. 2008). Allicin and diallyl disulfide at the levels of 0, 0.5, 5 and 10 mg/l medium were incubated using inoculum either from the sheep fed on high concentrate diet or high forage diets to study the interactions between additives and type of inoculum and found that the effects of allicin were more pronounced with the inoculum from sheep fed on high concentrate diet, whereas those of diallyl disulfide with the inoculum from high forage fed sheep (Kamel et al. 2003). They demonstrated the potential of these two compounds as rumen modifier but their stability is still doubtful. Wanapat et al. (2008) reported no effect on dry matter intake and nutrients apparent digestibility by garlic powder supplementation.

Garlic in livestock production

In livestock production industry (dairy and meat industry), use of antibiotics as growth promoters in animal diet is highly objectionable due to their residual effects. The European Union's Agricultural ministry has banned use of antibiotics in animal feed. Replacement of antibiotics with natural plant products, in which most of them are categorised as generally recognised as safe for human consumption (FDA 2004) is a highly acceptable approach. The plant products rich in secondary metabolites exhibit anti-microbial activity and therefore affect rumen microbial eco-system directly. The plants containing secondary metabolites can be screened for their potential to improve rumen fermentation and can be recommended as a feed additive to modify rumen fermentation for increased livestock productivity.

Cardozo et al. (2005) replaced antibiotic growth promoters with natural plant extracts for beef industry. The criteria used for selection was an increase or no effect on total VFA and decreased acetate:propionate ratio which reflects improved nutrient utilisation efficiency of the animal. It was found that ethanol extract of garlic (0.7% allicin) at the rate of 30 mg/l using rumen liquor from beef cattle fed on high concentrate diet could induce required changes in in vitro batch culture fermentation at pH 5.5, but at pH 7.0 the extract tended to decrease total VFA. Inclusion of garlic also resulted in lower ammonia nitrogen (which is again pH dependent) which might be due to decreased deamination or bacteria used peptides and amino acids as nitrogen source. Higher doses (300 mg/l) resulted in negative effects on both the pH, confirming anti-microbial activity of garlic. They concluded that garlic extract can be used with high concentrate diet in beef industry. The results from different laboratories revealed that there are many factors which determine the biological expression of essential oils like the type of oil, dose, diet of the animal. Since pH of the rumen depends on the diet and as most of the activity of plant secondary metabolites is pH dependent, the diet of animal seems to be an important factor. Therefore to elucidate the possibility of any plant containing secondary metabolites as a feed additive for manipulation of rumen metabolic process, all these factors have to be taken into consideration.

A commercial product having a mixture of garlic oil (G) and cinnamaldehyde (C) was tested in dairy and beef cattle. In dairy cattle, feeding of G+C resulted in high level of small peptides and amino acids and reduction in the levels of branched chain fatty acids and ammonia nitrogen. Total VFA was not affected but acetate:propionate ratio decreased showing better energy utilisation accompanied with 12% methane reduction. Milk production increased by 1.2 litre per day and milk protein also increased. The authors were of the opinion that the product equilibrate both protein and energy utilisation resulting in improved production. Addition of yucca extract or live yeasts (Saccharomyces cerevisiae) with G+C further enhanced the beneficiary effects (Kamel and Greathead 2008). But another study indicated that inclusion of garlic bulb or garlic husk at different levels in the diet of growing lambs did not affect growth performance, feed conversion ratio and carcass characteristics of the lambs (Bampidis et al. 2005). Jambrenghi et al. (2005) used garlic as a natural antioxidant and found that feeding of garlic extract was more effective than fresh garlic, in stabilising lipid oxidation in lamb meat. Lee et al. (2009) also demonstrated antioxidant activity of garlic and aged black garlic in mice. There was significant increase in antioxidant enzyme (superoxide dismutase and glutathione peroxidase) activities in the mice fed on garlic and aged black garlic supplemented diets, whereas catalase was increased only in black garlic fed mice. The black aged garlic was more potent antioxidant.

The reports from various laboratories as discussed previously, on garlic and its components, clearly indicate that there is a great potential in garlic to be used as a feed additive to manipulate rumen microbial eco-system for economic and eco-friendly livestock production. The use of garlic in the feed of animal is also safe as it is already in practice in human food. But before recommending it for practical application, there is a need for long-term feeding trials using variable doses, variable components with different feeding regimen and also frequency of feeding. Moreover, since the number of plants containing secondary metabolites have already been screened for their ability to manipulate rumen metabolism and each one of them has its own merits and demerits, it will be better if a mixture of garlic with other plants selected for their specific characteristics can be developed and used as a feed additive for manipulation of rumen microbial eco-system for improving livestock productivity.