Chemical Composition and in vitro and in vivo Digestibility of Tea Waste in Sheep

Document Type: Research Articles


1 Department of Animal Nutrition, Animal Science Research Institute of Iran (ASRI), Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran

2 Emam Khomeini Higher Education Center, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran


Tea processing includes several stages and in three of them namely drying, fiber-mat and screening some of the product is wasted. In this study, 25 percent of the tea processing plants in Iran were selected. Three samples were taken from each plant and analyzed for chemical composition and in vitro and in vivo digestibility and voluntary feed intake. Results showed that crude protein (CP) content of different tea by-products including drying, fiber-mat and screening were 198, 193 and 194 g/kg dry matter (DM), respectively, without significant difference among them. Concentration of ash were 64.0, 62.0 and 56.0 g/kg DM for drying, fiber-mat and screening respectively which was significantly (P<0.05) the lowest in Screening. The content of K, Ca, P, Mg and Na were 16.2, 4.2, 2.5, 2.2 and 0.73 g/kg DM, respectively, without significant difference among the samples. The mean values for in vitro digestibility coefficient of DM, organic matter (OM), OM/DM and gross energy of the tea wastes were 0.48, 0.44, 0.42 and 0.42, respectively. Inclusion of tea waste in the diet significantly (P<0.05) reduced in vivo digestibility coefficient of CP and neutral detergent fiber (NDF). The digestible energy was 10.0 MJ/kg which were not significantly different among the three types of tea waste. Inclusion of 80 g/kg DM tea waste in the diet significantly (P<0.05) reduced voluntary feed intake. Tea waste can be included in the diet of ruminants but its level should be limited due to presence of anti-nutritional factors.



The shortage of good quality feed has long been a serious constraint to the optimum efficiency of animal production in many parts of the world. Such a problem has been identified in Iran since the last four decades. While the shortage of feeds and fodder is well recognized, it is also equally important to understand how the available unconventional feed resources and by-products can be efficiently utilized. Over the two past decades, considerable emphasis has been placed on the beneficial role of agricultural wastes in livestock production in the country. By-products from tea (Camellia sinensis) processing are available in northern area of Iran and may be a potential roughage source in an amount of about 4000 metric ton annually, would be a valuable feed for livestock in that part of the country (Ananthasubramainam et al. 1978; Nikkhah and Hosseini, 1985). Tea processing involves several stages; namely drying, fiber-mat and screening, during which some by-product is generated. The protein content of this by-product is relatively high but its nutritive value is not well known. Tea waste could be considered as roughage, which is relatively rich in crude protein (Konwar et al. 1986; Sutrdhar, 1990) that may be comparable to several legume forages like alfalfa and clover, at least with respect to its crude protein content. However, its digestibility may be affected by the tannin-polyphenolic compound (Jayasuriya et al. 1978) that may reduce the nutritive value compared to many forage legumes. Considering the available technology of tea leaves harvesting and the processing systems, the chemical composition and nutritive value of tea wastes may be variable. Its protein content has been reported from 179 to 280 g/kg DM. It could be also a rich resource of some micro nutrients such as magnesium and copper (Fazaeli et al. 2000). The nutritive potential of tea waste in the diets of non-ruminants has been reviewed by some workers (Daimary et al. 1988; Das and Singh, 2004). However, few attempts have been made to determine the nutritive value of different types of such wastes, therefore its optimum utilization for ruminants is not well defined because of unclear understanding about potentiality of tea waste. When using new feed resources in feeding of animals, it is necessary to get sufficient information about its nutritive value. The aim of this study was to compare the chemical composition and in vitro digestibility of three types of tea waste and assess the effect of tea waste on in vitro and in vivo digestibility and voluntary feed intake in sheep.



Sampling method

Twenty of the national total of 80 tea processing plants facilities were selected as sampling units throughout the northern area of the Caspian Sea according to the systematic randomized sampling method (Bajpai, 2010). Each selected plant was visited during the working season (from May to August). Tea waste was obtained from the drying, fiber-mat and screening stages of processing at each processing plant were sampled and 60 samples (20´3=60) in total, were obtained. Therefore, one sample per type of waste-tea was obtained per factory. Samples were packed in 2-litre plastic containers and then were labelled. At the arrival at the lab, the plastic containers were opened and each sample was mixed. A sub-sample (about 20 g) was taken and milled, using laboratory 1-mm sieves mill, and used for chemical analysis and digestibility measurements.


Chemical analysis and energy determination

Samples were analyzed for dry matter (DM, ID number 934.01), organic matter (OM, ID number 967.05), ash (ID number 942.05), crude protein (CP=N×6.25, ID number 976.05) and ether extract (EE, ID number 920.39) according to AOAC (2000). Neutral detergent fiber (NDF) assayed and expressed inclusive of residual ash (Van Soest et al. 1991). Sodium sulphite and α-amylase were not used. The gross energy (GE) was determined by using Parr adiabatic calorimeter (model 1261, Parr instrument, USA).


Measurement of minerals 

Phosphorous was determined by wet digestion method using ammonium molybdate method followed by spectrophotometer measurement (ID number 965.17) (AOAC, 2000). A wet digestion method, with 15 mL of concentrated HNO3 and 7.5 mL of concentrated HCLO4, was carried out for sample preparation for Ca, K, Na, Mg, Fe, Mn, Zn and Cu followed by atomic absorption spectrophotometric measurement (AOAC, 2000).


Measurement of total extractable phenolics and total tannins

Determination of total extractable phenolic compounds and total tannins was accomplished according to the method described by Makkar et al. (1993).


In vitro digestibility

The digestibility of samples for DM, OM and GE were determined by the in vitro system, using acid pepsin two-stage method (Tilley and Terry, 1963) and rumen liquor was collected from two fistulated mature cattle fed wheat straw-alfalfa based diet.


In vitro gas production technique

Samples (200 mg) were incubated in triplicate in 100 ml calibrated glass syringes according to the method described by Menke and Steingass (1988). Rumen liquor was collected from the three fistulated local breeds (Taleshi breed) of cattle fed at maintenance (8.65 MJ/kg ME and 102 g CP/kg DM). Diet contained 300 g/kg alfalfa, 350 g/kg wheat straw, 200 g/kg wheat bran, 145 g/kg barley grain, 2.0 g/kg vitamin and mineral supplement, 3.0 g/kg salt on dry matter basis. Gas production was recored at 2, 4, 6, 8, 12, 24, 48, 72 and 96 hours after incubation. The adverse effect of tannins on in vitro gas production of tea waste was assessed by adding of polyethylene glycol (PEG) to feed and gas production was measured. Cumulative gas production data were fitted to the exponential equation: Y= b(1-e-ct) of Orskov and McDonald (1979) modified by Blummel and Becker (1997). The ME content was calculated using equations of Menke and Steingass (1988) as:

ME (MJ/kg DM)= 2.20 + 0.136 Gp + 0.0057 CP + 0.00029 CP2


Gp: net gas production (mL) from 200 mg after 24 h of incubation and CP is crude protein in g/100 g DM.


Determination of voluntary feed intake and in vivo digestibility

Since the chemical composition of the three types of tea wastes were more or less similar and the little differences in concentrations of ash and minerals was unlikely to affect digestibility and feed intake, and on the other hand, in the processing plants the three types of residues are mix, a mixture was prepared by mixing equal proportions of drying, fiber-mat and screening tea waste. Tea waste was added at different levels to alfalfa to obtain five diets containing 0, 40, 80, 120 and 160 g/kg DM tea waste. Two groups of Afshari male sheep (n=5/group) were used for determination of voluntary feed intake and in vivo digestibility. Animals were housed in individual metabolic cages and fed the experimental diets containing 5 levels of tea waste in 5 successive periods. In the voluntary intake trial, animals (53±2.5 kg) received the diet 10% above appetite and the amount of feed and refused were recorded daily. In the digestibility trial, animals (51±1.7 kg) were fed at maintenance requirements and the amounts of feed offered and refused were recorded daily. Feces were collected, weighed and a sample (10% of total) was dried. Animals in both trials were fed twice daily at 8.00 and 15.00. Animals had free access to multi-mineral salt lick and water. Animals had one week break out of the metabolic cage before starting the next period.


Statistical analysis

A completely randomized design with 20 replications was used to assess the differences in chemical composition and in vitro digestibility of tea waste. A completely randomized design with 5 replications was used to assess the differences in in vivo digestibility and voluntary feed intake. Analysis of all data was carried out using general linear model procedure of SAS (1996). The difference between the means was determined using Duncan's multiple range test at P < 0.05.



Chemical composition

The only significant difference (P<0.05) in chemical composition including CP, NDF, EE and Ash among the three tea wastes (Drying, Fiber-mat and Screening) was observed in ash concentration (Table 1). No significant difference was observed in concentration of macro-minerals except for mg among the three tea wastes investigated in this study. Among the four measured micro-minerals (Table 1), manganese was not different among the three types of tea wastes while a significant difference (P<0.05) was observed between the concentration of iron, magnesium, zinc and copper among them.


In vitro and in vivo digestibility

No significant difference was observed among the three types of tea wastes for in vitro digestibility, organic matter (OM), organic matter in dry matter (OM/DM) and gross energy (GE). No significant change was observed in in vivo digestibility coefficient of dry matter, organic matter and organic matter in dry matter of alfalfa with increasing tea waste inclusion levels while, digestibility of NDF and especially crude protein was significantly reduced (P<0.05) at 160 g/kg DM substitution of tea waste (Table 4). Results of chemical composition and in vitro gas production from alfalfa and tea waste are presented in Table 3. In vitro gas production of tea waste after 96 hours incubation was less compared to alfalfa despite containing more CP and less NDF. On the other hand, total extractable phenolics and total tannin in tea waste was higher than that in alfalfa (Table 3). Potential gas production from alfalfa, tea waste and tea waste + polyethylene glycol (PEG) revealed significant difference in bioavailability of the three samples for rumen microorganisms (Figure 1).


Voluntary feed intake

The highest level of dry matter and organic matter intake was from alfalfa hay (P<0.05). No effect was observed by inclusion of 40 g/kg DM tea waste in diet but all other levels negatively affected intake as compared with control and the lowest intake was observed when alfalfa was substituted for tea waste at the level of 160 g/kg DM (Table 5).


Chemical composition

The three types of tea waste contained high dry matter (see Table 1). The advantage of such dry matter is the ease of separation of wastes from tea and its preservation. The range of crude protein content in the three tea wastes confirmed their high protein content. The values of CP for tea waste in this study were lower than 283 and 276 g/kg DM reported by Kondo et al. (2014) and Makoto et al. (2014), respectively. The protein content of tea wastes in this study was in agreement with data reported by Nikkhah and Hosseini (1985) (183.9 g/kg DM) and Kadirvel (1979) (192.0 g/kg DM). The values for ash obtained in this study was lower than those reported by Nikkhah and Hosseini (1985) and Konwar et al. (1986) who mentioned 76.0 and 91.0 g/kg DM, respectively, but were higher than the value (43.0 g/kg DM) reported by Kadirvel (1979).


Table 1 Chemical composition and gross energy content of tea processing residues (mean±Sd)


The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SD: standard deviation.

SEM: standard error of the means.


Table 2 The in vitro digestibility coefficient of tea processing residues (mean±Sd)


DM: dry matter; OM: organic matter and GE: gross energy.

SD: standard deviation.

SEM: standard error of the means.


Table 3 Chemical composition, in vitro gas production and metabolizable energy (ME) of alfalfa and tea waste used in in vivo digestibility trial (DM basis)


1 GP: in vitro gas production after 96h of incubation.

2 ME: metabolizable energy calculated using the equation: ME= 2.2 + 0.136 G + 0.0057 CP + 0.0002589 CP2 (Menke and Steingass, 1988).

CP: crude protein; NDF: neutral detergent fiber; EE: Ether extract; TEP: total extractable phenolics and TT: total tannins.


The ash content of tea wastes in this study was in the line with the finding of Ramdani et al. (2013) (61.4 g/kg DM). The values of Mn, Fe, Cu and Zn in this study were higher than those reported by Ramdani et al. (2013) (527, 116, 23.8 and 21.7 mg/kg DM, respectively). Some factors can affect the chemical composition of tea waste including: Tea cultivar, region, harvesting time and method of tea processing (Fazaeli et al. 2000), which may be some of the reasons for the findings of this study.


In vitro and in vivo assays

Non significant difference between the in vitro digestibility coefficients (Table 3) was expectable because most of chemical compositions of the three tea wastes were not significantly different (Table 1). The diets used in in vivo trials contained various levels of alfalfa and tea waste. Regarding lower NDF and ash content and also higher concentration of CP in tea waste as compared to alfalfa (Table 3), it was expected that nutritive value of tea waste would be higher but in vitro gas production from tea waste was lower than that of alfalfa. This implies that this feed may contain anti-nutritional factors that negatively affected rumen microbes (or rumen microbes are less capable of degrading tea waste). Content of total extractable phenolics and total tannins in tea waste was almost ten times higher than that of alfalfa (Table 3).


Table 4 Effect of different levels of tea waste on in vivo digestibility coefficient of diet, digestible energy and metabolizable energy


1 Calculated using the equation metabolizable energy (ME)= DE × 0.82 (Robbins, 1993).

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.


Figure 1 In vitro gas production from alfalfa, tea waste and tea waste + polyethylene glycol



Table 5 Voluntary feed intake of experimental diets in sheep


The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.


Concentration of total extractable phenolic compounds and total tannins in tea waste in this study were in agreement with the result of Torbati-Nejad (1988), Jayasuriya et al. (1978) and Konwar et al. (1992). Addition of PEG to tea waste significantly increased in vitro gas production. Affinity of PEG to tannins has been well documented (Makkar et al. 1995a). Improvement in in vitro gas production by addition of PEG indicates that PEG has inactivated some of anti-nutritional poly-phenolics compounds (Robbins et al. 1987; Reed, 1995; Silanikove et al. 2001). Reduction in digestibility coefficient of NDF and CP (Table 4) is very likely due to presence of inhibitory compounds specially tannins in tea waste (Torbati-Nejad, 1988; Jayasuriya et al. 1978; Konwar et al. 1992). Tannins can bind to the cell wall and reduce cell wall digestibility and consequently reduce production of volatile fatty acids (Makkar et al. 1995b; Robbins et al. 1987; Silanikove et al. 2001). Tannins may have positive effect through binding to feed proteins and make insoluble complexes and reduce digestibility in the rumen (Giner-Chavez, 1996; Kumar and Vaithiyanathan, 1990) and afterward, the low pH in the abomasums as well as in the small intestine can stimulate dissociation (Perez-Maldonado et al. 1995; Zahedifar et al. 2001). In this way, tannins can improve the total availability of protein in forages by reducing the wasteful deamination of protein in the rumen in diets where rumen degradable protein is in excess of microbial requirements (Mangan, 1988; Perez-Maldonado and Norton, 1996). It has been reported that tannins can bind to bacterial enzymes in the rumen and reduce efficiency of hydrolytic enzymes (Makkar et al. 1995b; Bhatta et al. 2000; Imik and Seker, 1999; Barry and Manley, 1984). It seems that apart from tannins, other compounds like caffeine are also responsible in this respect (Giner-Chavez, 1996).


Voluntary feed intake

A declining trend was observed in dry matter and other feed intake characteristics by substitution of alfalfa with tea waste in the diet. Torbati-Nejad (1988) reported that daily feed intake in sheep was 827 g when 250 g/kg DM of alfalfa was replaced with tea waste which was higher than the values observed in this study (Table 5). In another experiment inclusion of 100 g/kg DM tea waste in diet of fattening male calves did not affect intake (Nikkhah and Hosseini, 1985). Such difference is likely related to chemical composition and quality of tea waste. Quality of some agricultural by products and residues of feed processing plants could be very heterogeneous and their composition is dependent on the method of harvesting and processing (Fazaeli et al. 2000). Variation in ratio of leaf to stem and thickness of stem in tea waste affect ratio of fibrous fraction and concentration of anti-nutritional factors and consequently affect digestibility and palatability of feed (Landau et al. 2000; Silanikove et al. 1994; Silanikove et al. 2001). Astringency of feed which is related to content of condensed tannins and its protein precipitating capacity is an important factor affecting palatability of feed (Bajec and Pickering, 2008; Barahona et al. 1997).



Results of this study showed that chemical composition of residues of tea processing including drying, Fiber-mat and Screening was more or less similar. Tea by-product can be classified as low quality roughage. Presence of tannins in tea by-product limits availability of its nutrients especially protein therefore, it is suggested to include up to 4 percent of it in diet of sheep.



The authors wish to thank the Animal Science Research Institute of Iran for accepting and supporting this project.

Ananthasubramainam C.R., Menachery M., Devasia P.A. and Nair A.M.C. (1978). Effect of tea waste on growth in calves. Kerala J. Vet. Sci. 11(2), 185-191.

AOAC. (2000). Official Methods of Analysis. 17th Ed. Association of Official Analytical Chemists, Arlington, Washington, DC., USA.

Bajec M.R. and Pickering G.J. (2008). Astringency: Mechanisms and Perception. Crit. Rev. Food Sci. Nutr. 48, 1-18.

Bajpai N. (2010). Business Statistics. Pearson Education India, Gwalior, India.

Barahona R., Lascano C.E., Cochran R., Morrill J. and Titgemeyer E.C. (1997). Intake, digestion, and nitrogen utilization by sheep fed tropical legumes with contrasting tannin concentration and astringency. J. Anim. Sci. 75, 1633-1640.

Barry T.N. and Manley T.R. (1984). The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. Br. J. Nutr. 51, 493-504.

Bhatta R., Krishnamoorthly U. and Mohammed F. (2000). Effect of feeding tamarind (Tamarindus indica) seed husk as a source of tannin on DMI, digestibility of nutrients and production performance of crossbred cows in mid-lactation. Anim. Feed Sci. Technol. 83, 67-74.

Daimary P., Saikia A. and Baruah K.K. (1988). Possibility of including decaffeinated tea waste as a source of protein. Indian J. Anim. Prod. Manage. 4(1), 11-15.

Das H. and Singh S.K. (2004). Useful by-products from cellulosic wastes of agricultural and food industry-a critical appraisal. Crit. Rev. Food Sci. Nutr. 44, 77-89.

Fazaeli H., Nik-Khah A. and Mirhadi S.A. (2000). Determination of chemical composition and gross energy of feed resources in Gilan province of Iran. Anim. Sci. J. (Pajouhesh and Sazandegi). 46, 100-105.

Giner-Chavez B.I. (1996). Condensed tannins in tropical forages. Ph D. Thesis. Cornell Univ., Itaca, New York.

Imik H. and Seker E. (1999). Effects of different tannin sources on feed consumption, live weight gain, mohair production and quality of Angora goats. Lalahan Hayvanc. Arast. Enst. Derg. 39(1), 85-100.

Jayasuriya M.C.N., Panditharatne S. and Roberts G. (1978). Spent tea leaf as a ruminant feed. Anim. Feed Sci. Technol. 7(2), 219-226.

Kadirvel R. (1979). Spent tea waste dust as an ingredient for poultry. Indian J. Poult. Sci. 14(4), 194-200.

Kondo M., Hirano Y., Kita K., Jayanegara A. and Yokota H. (2014). Fermentation characteristics, tannin contents and in vitro ruminal degradation of green tea and black tea by-products ensiled at different temperatures. Asian-Australasian J. Anim. Sci27, 937-945. 

Konwar B.K., Ahmed H.F., Phukan B. and Medhi A.K. (1992). Utilization of decaffeinated tea waste in crossbred calves. Indian Vet. J. 69, 25-28.

Konwar B.K., Mehdi A.K., Das A.K. and Phukan B. (1986). Studies on the effect of feeding decaffeinated tea waste in broilers. Indian J. Poult. Sci. 21(1), 11-15.

Kumar R. and Vaithiyanathan S. (1990). Occurrence, nutritional significance and effect on animal productivity of tannins in tree leaves. Anim. Feed Sci. Technol. 30, 21-38.

Landau S., Silanikov N., Nitsan Z., Barkai D., Baram H., Proviza F.D. and Perevolotsky A. (2000). Short-term changes in eating patterns explain the effects of condensed tannins on feed intake in heifers. Appl. Anim. Behav. Sci. 69, 199-213.

Makkar H.P.S., Bluemmel M., Borowy N.K. and Becker K. (1993). Gravimetric determination of tannins and their correlations with chemical and protein precipitation methods. J. Sci. Food Agric. 61, 161-165.

Makkar H.P.S., Blummel M. and Becher K. (1995a). Formation of complexes between polyvinylpolypyrrolidones or polyethylene glycols and tannins and their implication in gas production and true digestibility in vitro techniques. Br. J. Nutr. 73, 897-913.

Makkar H.P.S., Blümmel M. and Becke, K. (1995b). In vitro effects of and interactions between tannins and saponins and fate of tannins in the rumen. J. Sci. Food Agric. 69, 481-493.

Makoto K., Yoshiaki H., Kazumi K., Anuraga J. and Hiro-omi Y. (2014). Fermentation characteristics, tannin contents and in vitro ruminal degradation of green tea and black tea by-products ensiled at different temperatures. Asian-Australasian J. Anim. Sci. 27(7), 937-945.

Mangan J.L. (1988). Nutritional effect of tannins in animal feeds. Nutr. Res. Rev. 1, 209-231.

Menke K.H. and Steingass H. (1988). Estimation of the energetic feed value from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28, 7-55.

Nikkhah A. and Hosseini J. (1985). Utilization of tea waste in the nutrition of finishing calves. Iranian J. Agric. Sci. 16, 43-50.

Orskov E.R. and McDonald I. (1979). The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92, 499-503.

Perez-Maldonado R.A. and Norton B.W. (1996). The effect of condensed tannins from Desmodium intortum and Calliandra calothyrsus on protein and carbohydrate digestion in sheep and goats. Br. J. Nutr. 76, 515-533.

Perez-Maldonado R.A., Norton B.W. and Kerven G.L. (1995). Factors affecting in vitro formation of tannin protein complexes. J. Sci. Food Agric. 69, 291-298.

Ramdani D., Chaudhry A.S. and Seal C.J. (2013). Chemical composition, plant secondary metabolites, and minerals of green and black teas and the effect of different tea-to-water ratios during their extraction on the composition of their spent leaves as potential additives for ruminants. J. Agric. Food Chem. 61, 4961-4967.

Reed J.D. (1995). Nutritional toxicology of tannins and related polyphenols in forage legumes. J. Anim. Sci. 73, 1516-1528.

Robbins C.T. (1993). Wildlife Feeding and Nutrition. California Academic Press, San Diego, USA.     

Robbins C.T., Harley T.A., Hagerman A.E., Hjeljord O., Baker D.L., Scwartz C.C. and Moutz W.W. (1987). Role of tannins in defending plant against ruminants: Reduction in protein availability. Ecology. 68, 98-107.

Silanikove N., Nistan Z. and Pervolotsky A. (1994). Effect of polyethylene glycol supplementation on intake and digestion of tannin-containing leaves (Ceratenia siliqua) by sheep. J. Agric. Food Chem. 42, 2844-2847.

Silanikove N., Pervolotsky A. and Provenza F.D. (2001). Use of tannin-binding chemicals to assay for tannins and their negative posting digestive effects in ruminants. Anim. Feed Sci. Technol. 91, 69-81.

SAS Institute. (1996). SAS®/STAT Software, Release 6.11. SAS Institute, Inc., Cary, NC. USA.

Sutrdhar P.R. (1990). Study on the utilization of spent tea dust as feed for broilers. MS Thesis. Bangladesh Agricultural Univ., Mymensigh, Bangladesh.

Tilley J.M.A. and Terry R.A. (1963). A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassland Soc. 18, 104-111.

Torbati-Nejad N. (1988). Nutritive value of wheat straw, barley straw, rice straw, rice hull and tea waste. MS Thesis. University of Tarbiat Modarres, Tehran, Iran.

Van Soest P.J., Robertson J.B. and Lewis B.A. (1991). Methods for dietary fiber neutral detergent fiber and non-starch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597.

Zahedifar M., Castro F.B. and Orskov E.R. (2001). Effect of hydrolytic lignin on formation of protein-lignin complexes and protein degradation by rumen microbes. Anim. Feed Sci. Technol. 95, 83-92.