ST2B-2-2: Potential Use of Paper Mulberry Leaves for Silage Production

Silage is an important ruminant feed produced by an anaerobic fermentation process of lactic acid bacteria to preserve nutritional values of animal forage. Silage production can be adapted to many animal crops and also some agricultural wastes of high nutritional contents. In this study, a preliminary trial on making well preserved and high nutritional silage of paper mulberry (Broussonetia papyrifera) leaves, by-product (protein content ≈ 20% dry basis) of hand papermaking industries was obtained at a laboratory scale in 25-ml microsilos by a natural ensiling process of indigenous microflora naturally present in the plants. During ensiling process, the loads of lactic acid bacteria initially increased within a day and then decreased after seven days according to a drop in pH to 5.8 at the end of ensilage (40 days). Growths of enterobacteria being capable to cause detrimental impacts on silage qualities were inhibited by produced lactic acid. Nutritional values of leaves were highly preserved as only 9.5% protein was lost and low ammonia nitrogen produced (4%). However, lactic acid contents were not efficiently produced and the pH of ensiled products was still too high since the leaves contained a low content of water soluble carbohydrates. A manipulating ensilage was then carried out by the addition of sucrose (1% of fresh weight) to improve the fermentation efficiency. The results suggested a higher amount of lactic acid produced by 70% while leaf proteins were still conserved. INTRODUCTION The fresh forage of grass and legume is recognized as the best cattle feed since it contains high nutritional values and desirable tastes. The use of the fresh forage is more favorable but it is Kasetsart Agriculture and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, Bangkok 10900, Thailand Department of Biotechnology Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand Final Report The Research Project for Higher Utilization of Forestry and Agricultural Plant Materials in Thailand (HUFA) 772 seasonally available. Occasionally, the farmers do not have sufficient fresh materials to use. Hence, they learn to harvest the fresh forage at peak nutritional content and preserve by different methods to ensure the longer shelf-life and yearly availability of animal fodder. The most simple and popular method is a field-drying (sun drying) method for making hays. Artificial drying method is also applied for hay produciton in some certain areas. Recently, a preservation of forage crops by means of microbial fermentation to produce silage attains more interest. A production of ensiled feed has several advantages over making hay. To produce hay, the fresh forage is usually harvested during the rainy season, drying is, therefore, slow and difficult (Meeske et al., 1999). Besides, improvements in technology and a better understanding of the fermentation process help promoting silage production. Ensilage, a microbial fermentation process of conserving plant nutritional and energetic values, can be adapted to many crops grown as animal fodder including the foliage of corn, oats, barley, grain sorghum, and similar crops. Furthermore, preserved silage can be made from forage grasses, alfalfa and other legumes used for making hay. Regardless of raw materials to be used, ensiling is objected to develop lactic acid rapidly and sufficiently to suppress putrefaction and able to store nutritional-preserved feed until needed. The normal practice of ensiling precess begins with the harvest of forage having high nutultive values and dry matter digestibility. Harvested forage is then chopped into small pieces and rapidly packed into a close container called silo. During ensiling process, a fermentation process by natural microflora can take place. It is necessary to maintain anaerobic conditions to favorably and rapidly promote lactic acid production by lactic acid bacteria and lower the pH to around 4.0 for preserving nutritional fodder. An increased ensiling rate and improved silage quality can be achieved with suitable microorganisms (i.e. lactic acid bacteria) and substrates (i.e. moisture and nutrition content) used under a suitable and controlled environment. Accordingly, rather to be produced by natural fermentation, silage can be obtained by an artificial fermentation. This can be manipulated by the addition of many additives including water soluble carbohydrates (molasses, glucose), bacterial inoculum, acids and acid salts (Meeske et al., 1999; Fenlon et al., 1995: Padmaja and Balagopalan, 1991; Alli et al., 1984; Ely et al., 1981). Paper mulberry (Broussonetia papyrifera Linn.) is a regional plant typically found in north Thailand. Its bark is recognized as one of the most important raw materials for hand papermaking. Final Report The Research Project for Higher Utilization of Forestry and Agricultural Plant Materials in Thailand (HUFA) 773 Mulberry hand-made paper is very attractive and popular for making decorative products such as flowers, book-mark, cards and photo frames. After 1-year old, the stem of mature plant is cut and disbarked; the bark is then used for papermaking. Most of mulberry leaves of collected stems are discarded as agricultural waste. The leaves, in fact, contain a high protein content upto 20% dry basis and are still under utilized of value nutrients. This preliminary work was aimed to investigate a potential of producing well-preserved silage from paper mulberry leaves by indigenous microflora naturally present on the standing plant. Changes with respect to the microbial and biochemical properties during ensiling process were also documented along with the evaluation of nutritive values and quality of the silage. MATERIALS AND METHODS Materials Paper mulberry leaves of 1-year old plants were collected from different local fields in north Thailand. Properties of paper mulberry leaves Protein content in paper mulberry leaves were quantified by measuring total Kjeldahl nitrogen using a factor of 6.25 as described by AOAC methods (AOAC, 1990). Water soluble carbohydrate (WSC) contents in tubers were quantified as followed by Gaosong et al. (1997). Leaves of 10 g were extracted with 30 ml distilled water at 4°C overnight with a constant shaking of 150 rpm. The mixture was then filtered and the retentate was rewashed with 20 ml distilled water for three times. Collected filtrate was adjusted to 100 –ml volume and used for analyzing total sugar and reducing sugar contents by phenol-sulfuric method (Dubois, 1956) and the dinitrosalicylic acid method as described by Miller (1959). Ensiling A natural ensiling process of paper mulberry leaves was carried out in glass bottles with closed fitted covers as laboratory scale microsilos. Each 25 ml glass bottle was packed with 50 g of paper mulberry leaves chopped to about 1 – 2 cm in length. The moist leaves in the microsilo were Final Report The Research Project for Higher Utilization of Forestry and Agricultural Plant Materials in Thailand (HUFA) 774 pressed manually to remove the air from inter-particle spaces. Then the microsilos were incubated at 45°C for 40 days and the samples were taken at desired intervals. A manipulating ensilage of paper mulberry leaves was also conducted by the addition of sucrose (1% of fresh weight). The ensiling process was then similar to natural one described previously. Analysis Representative samples of fresh and ensiled paper mulberry leaves were analyzed microbially and biochemically. The total count of enterobacteria and lactic acid bacteria (LAB) were determined by preparing decimal dilutions in sterile 0.1% peptone then spreading on enterococci and MRS agar plates (Merck, Germany), respectively. All plates were incubated at 45°C for 48 h. Water soluble carbohydrate content (WSC) was estimated by using phenol sulfuric acid method (Dubosi et al., 1956). Reducing sugar content remaining in the silage was analyzed by using the dinitrosalicylic acid method as described by Miller (1959).