Bioinformatics study of phytase from Aspergillus niger for use as feed additive in livestock feed.

BACKGROUND: Phytase supplementation in rations can reduce their phytic acid composition in order to enhance their nutritional value. Aspergillus niger is a fungus that can encode phytase. This study aims to determine the characteristics of its DNA sequences and amino acid composition that encode the phytase enzyme, as well as to determine the primer designs. METHOD: This study used gene sequence data and protein-encoding phytase from Aspergillus niger that was collected manually from NCBI and PDB. The data was analyzed using SPDBV and then be aligned using the ClustalW Multiple Alignment features. The phylogenetic tree was built by Mega11 software. Primers were designed from selected candidate sequences that were analyzed. The designed primers were then simulated for PCR using FastPCR and SnapGene software. RESULTS: There are 18 Aspergillus niger phytases in NCBI which is 14.87% of the total Aspergillus. There are 14 Aspergillus niger phytases that have identity above 95%. Aspergillus niger 110. M94550.1 is the closest strain to the PDB template. Candidate sources of phytase genes are Aspergillus niger 110.M94550.1, 48.2.BCMY01000003.1, and 92.JQ654450.1. The primer design has 2 possibilities of self-annealing and high melting temperature on the reverse primer. PCR simulation shows that the primer design can attach completely but still has the possibility of mispriming. CONCLUSION: This study suggests promising results for the future development of phytase enzyme production from Aspergillus niger as a feed additive using genetic engineering to enhance the quality of livestock feed in Indonesia.

The effects of heat-moisture treatment on resistant starch levels in cassava and on fermentation, methanogenesis, and microbial populations in ruminants.

Background and Aim: Resistant starch (RS) is difficult to digest in the digestive tract. This study aimed to evaluate the effects of heat-moisture treatment (HMT) on RS in cassava and examined its impact on rumen fermentation. Materials and Methods: Cassava flour was used as a raw material and used in a randomized block design with four different cycles of HMT as the treatments and four different rumen incubations in vitro as blocks. Treatments included: HMT0: without HMT (control), HMT1: one HMT cycle, HMT2: two HMT cycles, and HMT3: three HMT cycles. Heat-moisture treatment processes were performed at 121°C for 15 min and then freezing at -20°C for 6 h. Analyzed HMT cassava starch characteristics included components, digestibility, and physicochemical properties. In in vitro rumen fermentation studies (48 h incubation) using HMT cassava, digestibility, gas production, methane, fermentation profiles, and microbial population assessments were performed. Results: Heat-moisture treatment significantly reduced (p < 0.05) starch, amylopectin, rapidly digestible starch (RDS), and slowly digestible starch levels. In contrast, amylose, reducing sugars, very RDS, RS, and protein digestion levels were significantly increased (p < 0.05). Additionally, a reduced crystallinity index and an increased amorphous index were observed in starch using Fourier-transform infrared analyses, while a change in crystalline type from type A to type B, along with a reduction in crystallinity degree, was observed in X-ray diffraction analyses. Heat-moisture treatment significantly (p < 0.05) reduced rumen dry matter (DM) degradation, gas production, methane (CH4 for 12 h), volatile fatty acid (VFA), and propionate levels. In addition, acetate, butyrate, and acetate/propionate ratios, as well as population of Streptococcus bovis and Bacteroides were significantly increased (p < 0.05). However, pH, ammonia, and organic matter digestibility were unaffected (p > 0.05) by HMT. Conclusion: Cassava HMT altered starch characteristics, significantly increased RS, which appeared to limit rumen digestion activity, decreased rumen DM degradation, gas production, VFAs, and CH4 production for 12 h, but increased S. bovis and Bacteroides levels.

A meta-analysis to observe silage microbiome differentiated by the use of inoculant and type of raw material.

Silage fermentation is naturally carried out by lactic acid bacteria (LAB) to mainly produce lactic acid (LA) and other organic acids as preservatives. Along with fermentation time, the growth of LAB will replace and suppress undesirable microorganisms. This meta-analysis study aimed to explore silage microbiome differentiated by LAB inoculants and type of raw materials. A total of 37 articles with 185 studies and 475 datasets were used for building up the meta-database. Data were subjected to the mixed model methodology. The parameters observed were silage quality and silage microbiome post-ensiling process. Results revealed that four bacterial genera along with Weissella dominated the post-ensiling process. The addition of lactic acid inoculants in the silage has increased the abundance of Lactobacillus spp. and decreased the Shannon index significantly. Moreover, the abundance of both L. plantarum and L. buchneri increased, and subsequently, Weissella, Pseudomonas, Proteobacteria, pH value, ammoniacal nitrogen (NH3-N), coliforms, and the yeasts were decreased significantly due to the addition of LAB inoculants in silage (p < 0.05). Environmental factors such as temperature affected the existence of Pseudomonas, Exiguobacterium, and Acinetobacter. However, the dry matter, LA, acetic acid (AA), the ratio of LA to AA, and the LAB population were enhanced significantly (p < 0.05). Among the LAB types, the lowest abundance of Pseudomonas was due to the LAB group, while the lowest abundance of Weissella and Proteobacteria was due to the addition of the combined LAB group. In conclusion, the addition of LAB is effectively enhancing the silage microbiome and silage quality by altering bacterial diversity and the metabolic products of the silage materials for safe preservation.

Changes in rumen fermentation and bacterial profiles after administering Lactiplantibacillus plantarum as a probiotic.

Background and Aim: Lactiplantibacillus plantarum is one of the lactic acid bacteria that is often used as probiotics. This study aimed to evaluate the effects of Lactiplantibacillus plantarum TSD10 as a probiotic on rumen fermentation and microbial population in Ongole breed cattle. Materials and Methods: This study adopted an experimental crossover design, using three-fistulated Ongole breed cattle. Treatments were as follows: T0, control without probiotic; T1, 10 mL probiotic/day; T2, 20 mL probiotic/day; and T3, 30 mL probiotic/day. The basal diet of the cattle comprised 70% concentrate: 30% elephant grass (Pennisetum purpureum). The concentration of probiotic used was 1.8 × 1010 colony-forming unit (CFU)/mL. Results: We observed significantly lower acetate production compared with control (64.12%), the lowest values being in the T3 group (55.53%). Contrarily, propionate production significantly increased from 18.67% (control) to 23.32% (T2). All treatments yielded significantly lower acetate-propionate ratios than control (3.44), with the lowest ratio in the T3 group (2.41). The protozoal number decreased on probiotic supplementation, with the lowest population recorded in the T2 group (5.65 log cells/mL). The population of specific rumen bacteria was estimated using a quantitative polymerase chain reaction. We found that the population of L. plantarum, Ruminococcus flavefaciens, and Treponema bryantii, did not change significantly on probiotic supplementation, While that of Ruminococcus albus increased significantly from 9.88 log CFU/mL in controls to 12.62 log CFU/mL in the T2 group. Conclusion: This study showed that the optimum dosage of L. plantarum TSD10 as a probiotic was 20 mL/day. The effect of L. plantarum as a probiotic on feed degradation in rumen was not evaluated in this experiment. Therefore, the effect of L. plantarum as a probiotic on feed degradation should be performed in further studies.

Divergence effects between dietary Acacia and Quebracho tannin extracts on nutrient utilization, performance, and methane emission of ruminants: A meta-analysis.

Extracts of Acacia and Quebracho have been used as a feed additive in ruminant diets; the effects, however, have been varied. This study used a meta-analysis approach to evaluate the use of those extracts on nutrient utilization, performance, and methane production of ruminants. A database was developed from 37 published papers comprising 152 dietary treatments. The result showed that a higher concentration of tannins was associated with a decrease (p < 0.05) in nutrient intake and digestibility. An increasing tannin concentration was negatively correlated with ammonia, acetic acid, and the ratio of acetic to propionic acid. Methane production decreased (p < 0.01) with the increasing tannin concentration. Nitrogen (N) balance parameters were not affected by the tannin concentrations, but fecal N excretion increased (p < 0.01) as the tannin concentration increased. The relationships between the Acacia and Quebracho and the changes in organic matter intake, milk fat concentration, butyric acid, valeric acid, and methane production were significantly different. In conclusion, it is possible to use both condensed tannins (CT) extracts as a methane emission mitigation without impairing the ruminant performance. Furthermore, the Quebracho showed more pronounced to decrease ruminal protein degradation and lower methane emission than the Acacia.

Health-Promoting Properties of Lactobacilli in Fermented Dairy Products.

Bacteria of the genus Lactobacillus have been employed in food fermentation for decades. Fermented dairy products, such as cheese and yogurt, are products of high value known as functional food and widely consumed due to their positive health impact. Fermentation was originally based on conversion of carbohydrate into organic acids, mostly lactic acid, intended to preserve nutrient in milk, but then it develops in other disclosure of capabilities associates with health benefit. It is expected that during the manufacture of fermented dairy products, some bioactive peptides from milk protein are released through proteolysis. Lactobacilli have been recognized and received increasing attention as probiotics by balancing gut microbial population. Information of molecular mechanisms of genome sequence focusing on the microbial that normally inhabit gut may explain as to how these bacteria positively give impact on improving host health. Recent post-biotics concept revealed that health benefit can also be associated after bacterial lysis. This mini review focuses on the contribution of lactobacilli in dairy fermentation with health-promoting properties on human health.

Fermentation Characteristics and Microbial Diversity of Tropical Grass-legumes Silages.

Calliandra calothyrsus preserved in silage is an alternative method for improving the crude protein content of feeds for sustainable ruminant production. The aim of this research was to evaluate the quality of silage which contained different levels of C. calothyrsus by examining the fermentation characteristics and microbial diversity. Silage was made in a completely randomized design consisting of five treatments with three replications i.e.: R0, Pennisetum purpureum 100%; R1, P. purpureum 75%+C. calothyrsus 25%;, R2, P. purpureum 50%+C. calothyrsus 50%; R3, P. purpureum 25%+C. calothyrsus 75%; and R4, C. calothyrsus 100%. All silages were prepared using plastic jar silos (600 g) and incubated at room temperature for 30 days. Silages were analyzed for fermentation characteristics and microbial diversity. Increased levels of C. calothyrsus in silage had a significant effect (p<0.01) on the fermentation characteristics. The microbial diversity index decreased and activity was inhibited with increasing levels of C. calothyrsus. The microbial community indicated that there was a population of Lactobacillus plantarum, L. casei, L. brevis, Lactococcus lactis, Chryseobacterium sp., and uncultured bacteria. The result confirmed that silage with a combination of grass and C. calothyrsus had good fermentation characteristics and microbial communities were dominated by L. plantarum.

New species of the genus Metschnikowia isolated from flowers in Indonesia, Metschnikowia cibodasensis sp. nov.

A novel species, Metschnikowia cibodasensis, is proposed to accommodate eight strains (ID03- 0093(T), ID03-0094, ID03-0095, ID03-0096, ID03-0097, ID03-0098, ID03-0099, and ID03-0109) isolated from flowers of Saurauia pendula, Berberis nepalensis, and Brunfelsia americana in Cibodas Botanical Garden, West Java, Indonesia. The type strain of M. cibodasensis is ID03- 0093(T) (= NBRC 101693(T) =UICC Y-335(T) = BTCC-Y25(T)). The common features of M. cibodasensis are a spherical to ellipsoidopedunculate shaped ascus, which contains one or two needleshaped ascospores, and lyse at maturity. Asci generally develop directly from vegetative cells but sometimes from chlamydospores. The neighbor-joining tree based on the D1/D2 domain of nuclear large subunit (nLSU) ribosomal DNA sequences strongly supports that M. cibodasensis (eight strains) and its closest teleomorphic species, M. reukaufii, are different species by a 100% bootstrap value. The type strain of M. cibodasensis, ID03-0093(T), differed from M. reukaufii NBRC 1679(T) by six nt (five substitutions and one deletion) in their D1/D2 region of nLSU rDNA, and by 18 nt (five deletions, four insertions, and nine substitutions) in their internal transcribed spacer regions of rDNA, respectively. Four strains representative of M. cibodasensis (ID03-0093(T), ID03-0095, ID03-0096, and ID03-0099) showed a mol% G+C content of 44.05 ± 0.25%, whereas that of M. reukaufii NBRC 1679(T) was 41.3%. The low value of DNADNA homology (5-16%) in four strains of M. cibodasensis and M. reukaufii NBRC 1679(T) strongly supported that these strains represent a distinct species.

Oleibacter marinus gen. nov., sp. nov., a bacterium that degrades petroleum aliphatic hydrocarbons in a tropical marine environment.

Three Gram-negative, motile, mesophilic, aerobic, rod-shaped bacterial strains, designated 2O1(T), 1O14 and 1O18, were isolated from Indonesian seawater after enrichment with crude oil and a continuous supply of supplemented seawater. The strains exhibited high n-alkane-degrading activity, which indicated that the strains were important degraders of petroleum aliphatic hydrocarbons in tropical marine environments. Phylogenetic analyses based on 16S rRNA gene sequences of members of the Gammaproteobacteria showed that the isolates formed a coherent and distinct cluster in a stable lineage containing Oceanobacter kriegii IFO 15467(T) (96.4-96.5 % 16S rRNA gene sequence similarity) and Thalassolituus oleivorans MIL-1(T). DNA G +C content was 53.0-53.1 mol%. The major fatty acids were C(16 : 0), C(16 : 1)ω7 and C(18 : 1)ω9 and the hydroxy fatty acids were C(12 : 0) 3-OH and C(10 : 0) 3-OH. The polar lipids were phosphatidylglycerol, a ninhydrin-positive phospholipid(s) and glycolipids. The major quinone was Q-9 (97-99 %), which distinguished the isolates from Oceanobacter kriegii NBRC 15467(T) (Q-8; 91 %). On the basis of phenotypic, genotypic and chemotaxonomic data, including DNA-DNA hybridization, the isolates represent a novel genus and species, for which the name Oleibacter marinus gen. nov., sp. nov. is proposed. The type strain of Oleibacter marinus is 2O1(T) (=NBRC 105760(T) =BTCC B-675(T)).

Actinophytocola timorensis sp. nov. and Actinophytocola corallina sp. nov., isolated from soil.

Two actinomycete strains, ID05-A0653(T) and ID06-A0464(T), were isolated from soils of West Timor and Lombok island, respectively, in Indonesia. 16S rRNA gene sequence analysis clearly demonstrated that the isolates belonged to the family Pseudonocardiaceae and were closely related to the genus Actinophytocola. Strains ID05-A0653(T) and ID06-A0464(T) exhibited 98.1 and 98.2 % 16S rRNA gene sequence similarity, respectively, with Actinophytocola oryzae GMKU 367(T). The isolates grew well on ISP media and produced white aerial mycelium. Short spore chains were formed directly on the substrate mycelium. The isolates contained meso-diaminopimelic acid, arabinose and galactose as cell-wall components, MK-9(H(4)) as the sole isoprenoid quinone, iso-C(16 : 0) as the major cellular fatty acid and phosphatidylethanolamine as the diagnostic polar lipid. The DNA G+C contents of strains ID05-A0653(T) and ID06-A0464(T) were 69.7 and 71.2 mol%, respectively. On the basis of phenotypic characteristics, DNA-DNA relatedness and 16S rRNA gene sequence comparisons, strains ID05-A0653(T) and ID06-A0464(T) each represent a novel species of the genus Actinophytocola, for which the names Actinophytocola timorensis sp. nov. (type strain ID05-A0653(T)  = BTCC B-673(T)  = NBRC 105524(T)) and Actinophytocola corallina sp. nov. (type strain ID06-A0464(T)  = BTCC B-674(T)  = NBRC 105525(T)) are proposed.

Importance of lactobacilli in food and feed biotechnology.

The genus Lactobacillus is a heterogeneous group of lactic acid bacteria (LAB) with important implications in food fermentation. The ability to colonize a variety of habitats is a direct consequence of the wide metabolic versatility of this group of LAB. Consequently, lactobacilli have been used for decades in food preservation, as starters for dairy products, fermented vegetables, fish and sausages as well as silage inoculants. Lactobacilli have also been proposed as probiotics and microbial cell factories for the production of nutraceuticals. However, a wide range of applications of lactobacilli in food biotechnology remains potential, whereas a number of important strains still need to be discovered and characterized. This article provides an overview of the taxonomy of lactobacilli and describes four of the most significant case studies on the application of this group of LAB in food and feed biotechnology, including their use as probiotics, dairy starters, silage inoculants, and microbial cell factories. The importance of access to and exchange of biological material within and between different strain collections as a crucial step in expanding the range of different biotechnological applications of lactobacilli is also emphasized.

Dietzia timorensis sp. nov., isolated from soil.

An actinomycete strain, ID05-A0528(T), was isolated using the SDS-yeast extract pre-treatment method from soil under mahogany (Swietenia mahogani) trees in West Timor, Indonesia, and was examined by using a polyphasic taxonomic approach. Chemotaxonomic and phylogenetic characterizations demonstrated that the novel strain belongs to the genus Dietzia. 16S rRNA gene sequencing studies showed that the strain was related to Dietzia cinnamea (97.2 %). Results of phenotypic and phylogenetic analyses determined that strain ID05-A0528(T) is different from the known species of the genus Dietzia. It is proposed that the isolate should be classified as a representative of a novel species of the genus Dietzia, with the name Dietzia timorensis sp. nov. The type strain is ID05-A0528(T) (=BTCC B-560(T) =NBRC 104184(T)).

Actinokineospora baliensis sp. nov., Actinokineospora cibodasensis sp. nov. and Actinokineospora cianjurensis sp. nov., isolated from soil and plant litter.

Six actinomycete strains isolated from soil and plant-litter samples in Indonesia were studied for their taxonomic position by using a polyphasic approach. Phylogenetically, all the strains were located in the broad cluster of the genus Actinokineospora. Chemotaxonomic data [cell-wall diamino acid, meso-diaminopimelic acid; cell-wall peptidoglycan, type III (A1γ); major sugars, galactose and arabinose; major menaquinone, MK-9(H4); major fatty acid, iso-C16:0; major phospholipid, phosphatidylethanolamine] supported the affiliation of all six strains to the genus Actinokineospora. The results of DNA-DNA hybridization with DNA from type strains of Actinokineospora species with validly published names revealed three DNA-DNA relatedness groups. Group I (ID03-0561(T)) showed low relatedness to the other strains studied. The three strains in group II (ID03-0784(T), ID03-0808 and ID03-0809) formed a group with high relatedness (98-100 %) and showed low relatedness to the other strains studied. The two strains in group III (ID03-0810(T) and ID03-0813) showed 58-68 % relatedness to Actinokineospora terrae NBRC 15668(T) and showed low relatedness (2-24 %) to the other strains studied. The description of three novel species is proposed: Actinokineospora baliensis sp. nov., for the single strain in group I (type strain ID03-0561(T) =BTCC B-554(T) =NBRC 104211(T)), Actinokineospora cibodasensis sp. nov., for the strains in group II (type strain ID03-0784(T) =BTCC B-555(T) =NBRC 104212(T)), and Actinokineospora cianjurensis sp. nov., for the strains in group III (type strain ID03-0810(T) =BTCC B-558(T) =NBRC 105526(T)).

Streptomyces baliensis sp. nov., isolated from Balinese soil.

The taxonomic positions of actinomycete strains ID03-0915T and ID03-0825, isolated from soil on the Indonesian island of Bali, were examined using a polyphasic taxonomic approach. The morphological and chemotaxonomic characteristics of these organisms are typical of the genus Streptomyces. Phylogenetic analyses performed using almost-complete 16S rRNA gene sequences demonstrated that the strains were closely related to Streptomyces glauciniger and Streptomyces lilacinus. However, DNA-DNA hybridization and phenotypic characteristics revealed that the strains differed from known Streptomyces species. Therefore, we conclude that strains ID03-0915T and ID03-0825 (=BTCC B-563) represent a novel species of the genus Streptomyces, for which we propose the name Streptomyces baliensis sp. nov. The type strain is strain ID03-0915T (=BTCC B-608T=NBRC 104276T).

Kozakia baliensis gen. nov., sp. nov., a novel acetic acid bacterium in the alpha-proteobacteria.

Four bacterial strains were isolated from palm brown sugar and ragi collected in Bali and Yogyakarta, Indonesia, by an enrichment culture approach for acetic acid bacteria. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates constituted a cluster separate from the genera Acetobacter, Gluconobacter, Acidomonas, Gluconacetobacter and Asaia with a high bootstrap value in a phylogenetic tree. The isolates had high values of DNA-DNA similarity (78-100%) between one another and low values of the similarity (7-25%) to the type strains of Acetobacter aceti, Gluconobacter oxydans, Gluconacetobacter liquefaciens and Asaia bogorensis. The DNA base composition of the isolates ranged from 56.8 to 57.2 mol% G+C with a range of 0-4 mol%. The major quinone was Q-10. The isolates oxidized acetate and lactate to carbon dioxide and water, but the activity was weak, as with strains of Asaia bogorensis. The isolates differed from Asaia bogorensis strains in phenotypic characteristics. The name Kozakia baliensis gen. nov., sp. nov., is proposed for the four isolates. Strain Yo-3T (= NRIC 0488T = JCM 11301T = IFO 16664T = DSM 14400T) was isolated from palm brown sugar collected in Bali, Indonesia, and was designated as the type strain.

Identification of acetic acid bacteria isolated from Indonesian sources, especially of isolates classified in the genus Gluconobacter.

Sixty-four strains of acetic acid bacteria were isolated from Indonesian sources such as fruits, flowers, and fermented foods by the enrichment culture at pH 3.5. Forty-five strains were routinely identified as Acetobacter strains because of their oxidation of acetate and lactate to carbon dioxide and water and their Q-9 isoprenolog, corresponding to 70% of all the 64 acetic acid bacteria isolated. Eight isolates were identified as Gluconacetobacter strains because of their oxidation of acetate and lactate and their Q-10 isoprenolog, occupying 13% of all the isolates. The remaining 11 isolates, accommodated in the genus Gluconobacter because of no oxidation of acetate and lactate and because of their Q-10 isoprenolog, accounted for 17% of all the isolates. They were divided into two groups based on DNA base compositions. One comprised the seven isolates, which had high G1C contents of DNA ranging from 60.3 to 63.5 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter oxydans at values of 64-94% of DNA relatedness. The other comprised the remaining four isolates, which had low G+C contents of DNA ranging from 57.5 to 57.7 mol% and of which DNAs hybridized with that of the type strain of Gluconobacter frateurii at values of 63-77% of DNA relatedness. The high values of DNA relatedness, 84 to 96%, were obtained between the type strains of Gluconobacter cerinus and Gluconobacter asaii.