In vitro fermentation characteristic and digestibility of dairy ration supplemented with Aspergillus oryzae inoculum

In vitro experiment has been carried out to study the effect of Aspergillus oryzae inoculum (AOI) supplementation on dairy cow rations. The observed effects include the digestibility of crude protein, NDF, dry matter and organic matter in the in vitro digestive system of the ration using rumen-buffer fluid mixture. The Experiment design using complete randomize design (CRD) consist of four treatments: Control (Commercial dairy feed), C2 (Commercial dairy feed + 2 g AOI), C4 (Commercial dairy feed + 4 g AOI) and C8 (Commercial dairy feed + 8 g AOI). The additional administration of 2, 4 and 8 grams of AOI/kg rations showed a significant difference (p<0.05) of the total volatile fatty acid content. However, the ratio of acetate/propionate (A/P) was reduced significantly. The highest increase in protein, fiber and dry matter digestibility was shown by C4 treatment (supplementation of 4 g AOI/kg ration). Based on the results of this in vitro study, the supplementation of AOI to dairy cow rations improves the digestibility of nutrients in feed.


Introduction
The use of antibiotics in livestock business is currently prohibited.Therefore, the search for active ingredients to replace antibiotics in feed is a continuous thing to do.Feeding with probiotics has been carried out a lot, as well as giving live microbes to feed or direct feeding microbial (DFM) is a common thing to do.DFM agents usually consist of mono or multi-microbes that improve livestock performance through a balance of microbiota in the digestive system [1].One practical example of using DFM is using culture products after fermentation by Aspergillus oryzae.This product is known as Aspergillus oryzae culture (AOC).Aspergillus oryzae culture has been widely used and reported for its use in ruminant farming as a feed additive [2][3][4].Feeding AOC to ruminant livestock was reported to improve the microbial composition in the rumen [5], also causing improvements in fiber digestibility [6].While its use in feeding dairy cows is reported to provide various results, including increasing milk production, feed efficiency and cows becoming more resistant to heat stress [7,8,9].Aspergillus oryzae is a filamentous non-pathogen fungus that can produce several enzymes depends on the different substrate type to grow these fungi.It was reported that solid substrate fermentation using A oryzae could increase the nutritional value of the substrate.It was also reported that fermented products lead to improvements in the digestive system, increasing the absorption of nutrients as well as increasing the synthesis of vitamins, reducing anti-nutrients and a better converting amino acids and lipids [10,11,12].It was also reported that A. oryzae can produce L-carnitine using a submerged fermentation procedure [13].The utilization of Aspergillus oryzae culture (AOC) has been widely used.AOC contains hypha and possibly AO spores along with the material used as a substrate for the growth of the fungi.Lubis, et al [14] use onggok (cassava waste after the starch from cassava is taken) as a substrate and using the whole product (5% and 10%) for sheep feeding.However, administering Aspergillus oryzae spores directly is something new, in this case the Aspergillus oryzae spore inoculum is acting like as Lactobaccillus probiotic.
A. oryzae is non-pathogenic fungi and is regarded as safe (GRAS) [15,16] and the A. oryzae has been used as an ingredient in many types of traditional food in Asian countries such as Indonesia, Thailand, China, and Japan.In Japan, A. oryzae is known as "koji mold" which is grown through fermentation using solid substrates to produce fermented foods such as rice wine (sake), soybean paste (miso), Japanese soy sauce (shoyu) and others [17].Adding AOI as a feed supplement or feed additive will cause improvements in the digestibility of feed ingredients and improvements in the rumen microbiota and improvements in rumen characteristics.In this manuscript, we reported our research of the effect of A. oryzae inoculum on the in vitro digestibility rate of rations for 24 hours and changes in rumen fluid characteristics.Measurements carried out were the determination of TVFA (total volatile fatty acids), individual VFA content, ammonia, and microbial crude protein content in the in vitro system.

Materials and methods
Animal ethics clearance was granted by the Animal Ethics Clearance Committee of the IAARD in 2019 when the research plan was submitted.The A. oryzae used to make the inoculum was A. oryzae GS66 which is a collection from Indonesian Research Institute for Animal Production (IRIAP). A. oryzae GS66 is a fungus isolated from decaying garlic seeds.All experiments related to animals were conducted based on the Guideline of Animal Ethics and Welfare (Balitbangtan/Balitnak/Rm/02/2019).

Substrate preparation
Super fluffy rice was used as the substrate.It was soaked overnight, drained and then steamed for 15 minutes over a boiling water.Cooling down to room temperature around 28-30℃.

Fungal Cultivation
The spore A. oryzae was prepared as followed: the single colony of fungal from the purification streak in a petri dish was remove and inoculate in a 10 mL of PDA (Potato Dextrose Agar BD Difco 213400) slant and incubated for 5 days.A 5-day old spore was then harvested using 10 mL saline sterile/slant.The spore suspension from several slants was combined and will be used to prepare AO inoculum.As much as 20 mL spore suspension was applied to 1 kg substrate; the average spore concentration was 9.6 × 10 7 ±3456 spores/mL.The rice substrate previously described was added spore suspension and mixed thoroughly and put in sterile plastic trays size 20x30 cm and covered with another sterile tray for 3 days incubation period at room temperature.The product was then cut into small cubes, dried under 60℃ until dry (between 2-3 days), ground after cooled.

In vitro Experiment
In vitro experiment conducted with four treatments: Control (C, Dried King Grass: Commercial feed concentrate for dairy), C2 (C + 2 g AOI), C4 (C + 4 g AOI) and C8 (C + 8 g AOI).The control feed consisted of king grass and commercial feed concentrate (1:1) DM basis and was applied for average daily ration intake for dairy cattle in the experimental station at IRIAP-Ciawi as much as 10 kg DM/day.In the lab-scale we used a mixer capacity of up to 5 kg.

The microbial rumen inoculum
The rumen fluid was taken from fistulated cow early in the morning before feeding.To preserve the anaerobic condition and the constant temperature (39°C), it was put into an air-tight thermal container and brought to the laboratory.As soon as arrived in the laboratory, an approximately150-g of the rumen fluid sample was put into a blender and mixed with 450 ml of warm distilled water (1:3).Sample was blended for 2 minutes while being saturated with CO2, then strained through four layers of linen, the pH was measured directly using a pH meter.As much as 5 mL of filtrated rumen fluid were pipetted into a 10 mL McCartney bottle containing 1 mL of meta-Phosphoric acid (Merck 100546) (25% wt/vol).The rumen fluid sample was then stored at−20°C for volatile fatty acid (VFA) analyses in the future.At the end of the incubation (48 h) period, the ruminal pH of each incubation bottle was recorded (see below).
Buffer mixture was a combination of 1330 mL buffer solution A (KH2PO4, 10.0 g/L; MgSO4.7 H2O, 0.5 g/L; NaCl, 0.5 g/L; CaCl2.2H2O,0.1 g/L; and urea, 0.5 g/L) was added to 266 ml of buffer solution B (Na2CO3 , 15.0 g/L and Na2S•7H2O, 1.0 g/L) in a 2000 mL pre-warmed Erlenmeyer.The final pH was 6.8.Warm rumen microbe inoculum (100 mL) was added to 400 mL warm buffer mixture (39°C) and mix under CO2 purge.Feed ration (500 mg) was weighed out into a 120 mL capacity incubation bottle and 75 mL rumen-buffer mixture was added, the bottle was then tightly sealed with a rubber stopper, and incubation was performed at 39°C for 48 hours.In this experiment, the gas volume produced during the incubation periods was determined.These measurements were taken after 24-hr incubation.After measuring the amount of gas produced in each bottle, the remaining fermented substrate was measured using a pH meter.Furthermore, the volatile fatty acids (VFAs) in each fermentation bottle were determined by using gas chromatography.For extraction and determination of VFA was followed the method of Li et al [15].The frozen sample in vitro rumen flui thawed at 4°C and centrifuged for 5 minutes at 2,500 × g.The supernatant was pipetted (5 mL) into a centrifuge tube containing 1 mL of 25% meta-Phosphoric acid.After 3 hours standing at 4°C and centrifuged at 3000 g for 10 min.after that, two-millilitres supernatant was re-centrifuge for 15 minutes at 12,000 × g and 4°C, passed through 0.45 μm organic membrane, then the filtrate (1 mL) was taken and mixed with 200 μL 1% crotonic acid.Gas chromatography was used to determine the VFA content in the in vitro sample.The crude protein, NDF, Dry Matter and Organic matter digestibility was measured using above in vitro procedure with 5 replicates.The 48 hours incubation of in vitro bottles were filtered, and the residue was used for crude protein, NDF, Dry Matter and Organic matter content.

Results and discussion
Feed ration and AOI product were subjected to chemicalnutrient composition analysis and, showed in Table 1.The gross energy of AOI was quite high when compared with feed ration, however the crude protein (CP)of AOI is 48.36%.Supplementation of feed ration for 2, 4 and 8 g AOI/ Kg will increase 0.9672, 1.9344 and 3.8688 g CP respectively.The VFA, Total VFA and pH during in vitro fermentation of feed ration is shown in Table 2 The total VFA contents showed a difference content significantly among different level of AOI added.The acetate, propionate, butyrate and total volatile fatty acids (TVFAs) shown affected by the level of AOI supplement.The content of acetate and the ratio of acetate:propionate (A:P) showed a significant difference, while the increase of TVFA, propionate, butyrate was clearly shown.The content of propionate was significantly higher in the C8. and the pH, and A/P were also affected by the level of AOI.From this experiment.supplementation with AOI provides an increase in DM digestibility.The increase in DM digestibility in treatments C4 and C8 reached 21.13 and 19.09%, respectively (see Table 3).This increase in DM digestibility can occur because A. oryzae is a mold that produces enzymes [16], such as cellulases xylanases, and various proteases.These enzymes can digest fiber and protein increasing microbial colonization [17] of feed ingredients in the rumen which in turn increases the rate of fiber digestion to increase during 24 hours of incubation.Sun et al. [18] stated that the addition of AOC to feed gave positive results on microbial protein content when given to dairy cows.The results of this experiment also obtained similarities, that the addition of AOI can provide positive conditions for the fermentation process that occurs in the rumen.So that the growth of beneficial bacteria increases and suppresses the growth of protozoa.This causes more rumen bacteria to survive so they can digest nutrients properly as well as increasing the digestibility of dry matter and neutral detergent fiber fractions.

Rumen fermentation
In the experiments that have been carried out, the addition of AOI to the feed mixture significantly increased the content of TVFA, including individual VFAs such as acetate, butyrate, iso and n-valerate.In addition, the ratio of acetate/propionate was lower in the C2 and C8 treatments.Together with the results of the digestibility analysis, the increase in the rate of digestibility of the rations during 24 hours of fermentation led to a higher increase in TVFA in feeds that were treated with the addition of AOI (2.4 and 8 grams/Kg).The same results also showed that the addition of AOI to feed had a positive effect on rumen TVFA content thus causing an increase in fermentation efficiency and producing better VFA [19,17].Jouany et al. [20] also stated that the addition of AOC to sheep feed decreased the production of acetic acid in the rumen, but increased the production of propionic and butyric acids.Isovaleric acid is an acid that characterizes the growth of fiber-digesting bacteria in the rumen.The increased content of this acid can improve the fiber-eating microbial colonies so that the digestibility of the dry matter becomes higher.
Cellulose enzyme activity was detected in AOI (Table 1) and affected the digestibility of fiber biomass types.Cellulosic biomass is converted through hydrolysis by microbial enzymes in the rumen into a source of bio-energy that can be achieved biologically and chemically.The rumen microbial consortium is an ecosystem that is very active in producing enzymes with a broad spectrum.This enzyme plays a role in breaking complex bonds in cellulose compounds.Incubation and fermentation for 48 hours resulted in a decrease in fibre biomass, dry matter, and NDF in the treatment with the addition of AOI compared to the control treatment.However, the digestibility of crude protein tends to remain stable.This is a good thing, where there will be protein availability at the next digestive site (post-rumen).It can also be suspected that AOI may produce more fiber-breaking enzymes than protein.
The degree of rumen acidity does not appear to be affected by the addition of AOI in the ration (can be seen in Table 1).Higginbotham et al. [6] and Fondevila et al. [16], also noted that the addition of AOC did not affect rumen acidity in dairy cows.Several articles by other researchers said that giving an additional 1-6 grams of AOC daily showed a slight increase in rumen pH [21] and a decrease in rumen pH was shown when adding AOC up to 27 g/day [22].

Conclusion
From this study, it can be concluded that the addition of 4 g AOI / kg ration can improve the digestibility of crude protein, fibre fraction, dry matter and organic matter of dairy cattle feed.Digestibility improvement of these nutrients will lead to better livestock growth and production.Giving AOI probiotics directly to livestock gives hope for better results, but this needs to be proven by giving them in direct feed trials on livestock (in vivo).It is hereby suggested that further experiments be carried out, namely giving AOI probiotics directly to livestock in vivo.

Table 1 .
Chemical / Nutrient Composition of Feed Ration* and Aspergillus oryzae inoculum (AOI) *Feed ration is a mixture of King grass and commercial feed concentrate 1:1, dry matter basis.

Table 2 .
VFA, Total VFA and pH during in vitro Fermentation of feed ration supplemented with AOI a-c Means within a row followed by different lower-case letters differ significantly from each other (P<0.05).TVFA, total volatile fatty acids.NH 3-N, ammonia-nitrogen.MCP, microbial crude protein

Table 3 .
Crude protein, NDF, DM and OM digestibility of Fermentation of feed ration supplemented with AOI