学位論文内容の要旨

(1)

氏名 AJMAL WALI 授与した学位博士

専攻分野の名称農学

学位授与番号博甲第６４２４号

学位授与の日付２０２１年３月２５日

学位授与の要件環境生命科学研究科農生命科学専攻

（学位規則第４条第１項該当）

学位論文の題目 Microbial fermentation and quality preservation of agro-industrial wet by-products

（食品及び醸造副産物の微生物発酵による品質保持に関する研究）

論文審査委員教授齋藤昇教授森田英利教授西野直樹准教授荒川健佑

学位論文内容の要旨

A large variety of wet by-products are produced by the agriculture-base, food, and beverage industries.

Although animal husbandry has successfully made use of these by-products as ingredients of animal feed, thermal drying requires huge cost and fuel energy; hence, wet by-products are often preserved under anaerobic fermentation in silos.

During the natural ensiling process, the epiphytic lactic acid bacteria (LAB) of pre-ensiled materials play important roles in the acidification of silages, resulting in inhibition the growth of undesirable microorganisms. When a silo is opened, however, anaerobic conditions are no longer maintained, and aerobic microbes have the opportunity to grow. Therefore, secured aerobic stability of silages can confer a substantial advantage to farmers and consumers. In this dissertation, three experiments were carried out to examine the dynamics of bacterial and fungal microbiota associated with soybean curd residue, total mixed ration, and whole crop corn ensiling.

In the first experiment, ensiling of wet soybean curd residue (SCR), a wet by-product generated during tofu processing, was examined. Laboratory-scale silos were prepared using wet soybean curd residue (SCR) obtained from two tofu factories (F1 and F2) mixed with or without added beet pulp (BP). Sealing was performed on the day of tofu production (prompt sealing [PS]) or 2 days after SCR was piled and unprocessed (delayed sealing [DS]). In PS silage, the abundances of typical lactic acid-producing bacteria, such as Lactobacillus spp., Pediococcus spp., and Streptococcus spp. reached >50%. In DS silage, Acinetobacter spp. were the most abundant in F1 products, whereas Bacillus spp. were the most abundant in long-stored F2 products. The fungal microbiota were highly diverse. Although Candida spp., Aspergillus spp., Cladosporium spp., Hannaella spp., and Wallemia spp. were found to be the most abundant fungal microbiota, no specific genera were associated with factory, sealing time, or fermentation products.

In the second experiment, the effect of different ambient temperatures on bacterial and fungal dynamics of total mixed ration (TMR) silage was examined. Two types of practical TMR mixture with different ingredients formulation were obtained from a feed company and stored for 14 and 60 days at temperatures of 10ôC, 25ôC, room (RT), and 40ôC.

Regardless of the storage period, all TMR silages had lactic acid as the predominant fermentation product and exhibited acceptable aerobic stability after silo opening. The abundance of Lactobacillus spp. increased regardless of storage period and temperature. In 40^oC-stored TMR silages, the abundance of Lactobacillus spp. decreased and those of Aneurinibacillus spp., Bacillus spp., and Brevibacillus spp. increased to as high as 40%. A large number of fungal genera were detected at >1% level. Diutina spp. were the most abundant (>30%) fungi in pre-ensiled TMR mixtures. Aspergillus spp. and Vishniacozyma spp. were the second most in pre-ensiled TMRs. Regardless of storage period and temperature, Kazachstania spp. were the most abundant fungi in TMR silages. The findings that typical spoilage genera such as Candida spp., Saccharomyces spp., and Kluyveromyces spp. were kept suppressed over the ensiling may account for high aerobic stability of TMR silage.

In third experiment, the potential of LAB species isolated from TMR silage was examined. Using the cultures of L. buchneri, L. casei, L. farciminis, L. parafarraginis, L. paralimentarius, L. plantarum, B. subtilis, and B. coagulans isolates, inoculation experiment was carried out using whole crop corn silage. Hetero-fermentative LAB species were effective to inhibit deterioration after silo opening and the ability of L. parafarraginis was shown greater than that of L.

buchneri. The association of multiple LAB species that have significant inhibitory activities may contribute substantial resistance of TMR silage on exposure to air.

From three experiments conducted in this study, the dynamics of bacterial and fungal microbiota associated with wet by-products ensiling has been clarified and several LAB species have been shown as new additions to silage LAB inoculants. Major findings in this study may contribute greatly to improved utilization of wet by-products in livestock industry in cool, temperate, and tropical countries.

(2)

論文審査結果の要旨

食品産業及び醸造産業で生じる副産物は，エコフィードとして飼料化及び再利用することが望ましい。化石燃料による乾燥は約20%のコスト増加につながるため，発酵貯蔵が飼料化の第一選択肢になる。本研究では，

食品及び醸造副産物の発酵貯蔵における細菌及び真菌叢をアンプリコン解析で調査した。

実験1では，トウフ粕を排出当日あるいは2日後に密封して6ヶ月間貯蔵した。材料のトウフ粕は細菌叢の半分以上がAcinetobacterであり，密封遅延によってその割合はさらに高まった。早期密封ではLactobacillusや Pediococcusが優勢となったが，密封遅延ではAcinetobacterの割合が継続して高かった。真菌叢は細菌叢より多 様であり，Cladosporium，Hannaellaといった牧草類では報告例のない菌種が多かった。

実験2では，トウフ粕の割合を20%とする混合発酵飼料（発酵TMR）を調製した。エコフィード材料は季節を問わず生じるため，貯蔵温度を10，25，室温及び40℃として，発酵生成物及び微生物叢の変化を調べた。TMR 材料にはAcinetobacterが約20%の割合で存在したが，Lactobacillusはそのおよそ2倍検出された。10℃では貯蔵初期にAcinetobacterが増加したが，2ヶ月後には他の貯蔵温度と同様にLactobacillusが優勢となった。40℃では貯蔵初期にLactobacillusが優勢したが，長期貯蔵では一部がBacillus，Brevibacillus等に置き換わった。真菌叢ではDiutina，Kazachstaniaといった牧草類では報告例のない菌種が優勢であり， Candida，Saccharomyces，

Kluyveromycesといった好気的変敗の起因菌は非常に少なかった。

実験2の発酵TMRから培養法で分離したLactobacillus及びBacillusを用いて，実験3でトウモロコシホールクロップへの添加試験を行った。L. buchneri，L. parafarraginis，L. paralimentariusといった複数のLactobacillusが好気的変敗を抑制する効果を示し，これらが相加・相乗的に作用して発酵TMRの貯蔵性を高めていると考えられた。

これらの結果は，食品及び醸造副産物の発酵貯蔵に関わる微生物叢が牧草類とは大きく異なることを意味しており，牧草類の貯蔵に広く応用できる微生物の分離源となりうることも示している。得られ知見及び成果は関連分野で十分な新規性及びオリジナリティがあるものと評価されており，WALI Ajmal氏は環境生命科学研究科の博士（農学）の学位を受ける資格があるものと判断した。