豆渣之發酵保存及其在乳牛飼糧應用之體外評估

Abstract

豆渣為豆漿加工過程中的副產物，豆渣乾物質中含有近 30% 的蛋白質，具有成為飼料中蛋白質原料之潛力，卻因水分含量高而難以保存和利用。因此，本研究目的在利用發酵法保存豆渣，以利用乳酸菌產生的乳酸降低豆渣 pH 值來延緩腐敗，並評估豆渣發酵成品應用在反芻動物餵飼之價值，以增加豆渣之利用性。 試驗第一部分以商用乳酸菌及酵母菌混合菌粉進行接種，以三種水分含量（60、70 與 80%）與接種濃度（106、107 與 108 CFU/g）進行四個不同時間點 （3、6、12 與 24 天）的先期組合評估，並分析發酵期間成品之 pH 值、氨態氮、乳酸及揮發性脂肪酸濃度，配合微生物計數來評估發酵品質，以評估適合之發酵條件。成品於開封時進行有氧穩定性試驗，最後以最佳條件進行小規模（20公斤）與大規模（80公斤）量產發酵。試驗結果發現豆渣發酵第 24 天時，pH 值在所有處理組中皆可降至 4.5以下，其中以 106 CFU/g 接種濃度搭配 80% 水分的組別，pH 值最低且乳酸含量顯著較高，因此選擇此條件進行放大規模發酵。小規模（20 公斤）量產發酵試驗結果顯示，第 2 週後成品 pH 值顯著下降，但乳酸、乙酸及粗蛋白質含量在各週無差異，但貯存第 6 及 8 週的 pH 值、氨及乙酸含量顯著提高，顯示在此條件下發酵後，維持品質穩定之天數仍有限制。好氣穩定性結果顯示，氨含量在開封後第 24 小時顯著高於第 12 小時，而 48 小時的總生菌數也顯著較高。大規模（80 公斤）量產試驗中，發酵 1 週後成品 pH 值即降到 4.5 以下，發酵成品化學成分在發酵開始後4週內無顯著差異。 第二部分試驗以固定乳酸菌接種濃度（106 CFU/g）並搭配不同水分條件發酵之成品，透過體外消化試驗來評估發酵豆渣於乳牛飼糧的利用性。試驗結果顯示，發酵豆渣與原料各組別在試管內乾物質消化率（in vitro true dry matter digestibility, IVTDMD）、中洗纖維消化率（in vitro neutral detergent fiber digestibility, IVNDFD）與總產氣量和未發酵豆渣無顯著差異，但發酵豆渣組可降解部分（degradable fraction）含量與體外發酵後氨含量則顯著較高。原位（in situ）試驗結果顯示發酵豆渣之有效降解率（effective degradability, ED）較低（52.82 - 58.78%），然而蛋白質中快速降解部分、降解速率（kd）、瘤胃中可降解蛋白（rumen degradable protein，RDP）與瘤胃中不可降解蛋白（rumen undegradable protein，RUP）數值並無顯著差異。進一步將新鮮豆渣與發酵豆渣取代飼糧中大豆粕成分之 25、50、75 與 100% ，進行體外消化結果顯示，當各配方含有相似之粗蛋白、能量、RDP 與 RUP下，各組在 IVTDMD 與 IVNDFD 無顯著差異。在體外產氣評估顯示，發酵豆渣取代豆粕 25%、50% 組的總產氣量與可降解部分顯著較高，和控制組最接近。原位試驗結果則顯示，各組混合飼糧的降解速率及 ED 皆無顯著差異。 綜合上述，以適當發酵條件保存豆渣可以降低 pH 值，延長可使用期限，且在放大製程後也能達到效果，並可維持豆渣的營養價值。在體外試驗中，以不同比例之發酵豆渣取代大豆粕後不會影響體外消化率，且瘤胃中降解速率及有效降解率也無差異，顯示發酵豆渣具有取代乳牛飼糧中豆粕的潛力，作為穩定的替代性飼糧蛋白質的來源。

Okara is a by-product of soymilk manufacture. It contains about 30% of crude protein and shows the potential to be a protein feedstuff. However, okara has difficulties in preservation and transportation due to its high moisture content. Therefore, the purpose of this research is to improve the preservation of okara through lactic acid bacteria fermentation and decrease the pH value by lactate. The feeding value of okara is also evaluated for future application in ruminant diets. In the first experiment, commercial lactic acid bacteria and yeast mixture product was used as a starter to ferment okara under different moisture content (80, 70, and 60%) and starter inoculation levels (108, 107, and 106 CFU/g) for 3, 6, 12, and 24 days. The pH value, ammonia, lactate, volatile fatty acid content, and microbial counts were analyzed to investigate the optimal fermentation condition. The aerobic stability after opening was also evaluated. Subsequently, the small-scale (20 kg) and large-scale (80 kg) okara fermentation was carried out under optimal fermentation conditions. The condition test results showed the pH value of all test groups was less than 4.5 on the 24th day of fermentation. Among all treatments, the okara fermented with 106 CFU/g inoculation level with 80% moisture condition had the lowest pH value and higher lactate, and this condition was chosen for scale-up fermentation. The results of the small-scale (20 kg) fermentation test indicated that after 2nd week of fermentation, the pH value decreased significantly. The content of lactate, ammonia, and acetate showed no difference during fermentation. However, in the 6th and 8th weeks, the pH value, ammonia, and acetate content were increased significantly. It implied that fermentation treatment still had limitations on maintaining quality in a longer preserved time. The aerobic stability showed that only the ammonia content was higher in the 24 hr after opening and the total bacteria count was higher in 48 hr. The large scale (80 kg) fermentation results showed that the pH value was decreased to less than 4.5 after 1 week of fermentation, and no difference was shown in the chemical composition of the fermented product within 4 weeks. In the second experiment, the fermented product (with 106 CFU/g inoculation level) with different moisture conditions was used to evaluate the utilization of fermented okara in the dairy cow diet through in vitro and in situ experiments. The results indicated that the in vitro true dry matter digestibility (IVTDMD), in vitro neutral detergent fiber digestibility (IVNDFD), and the in vitro gas production volume of each group had no differences, but fermented okara groups had higher degradable fraction (b) and ammonia content during in vitro rumen fluid fermentation experiment. The result of in situ tests showed that the effective degradability (ED) of fermented okara was lower (52.82 – 58.78%), but no significant difference was shown in the rapidly degradable fraction of protein, degradation rate (Kd), rumen degradable protein (RDP), and rumen undegradable protein (RUP). Finally, fresh okara and fermented okara (under 106 CFU/g inoculation level, 80% moisture, fermented for 24 days) were applied to replace 25, 50, 75, and 100% of the soybean meal in the total mixed ration (TMR) of dairy cows. All test TMRs contained similar crude protein, energy, RDP, and RUP level. The results showed no differences in IVTDMD and IVNDFD among all test TMRs. The TMR with fermented okara in 25 and 50% replacement ratios had higher in vitro gas production volume and soluble fraction, and the value was similar to the control group. The result of the in situ tests also indicated that the degradation rate and ED of all groups were similar. In conclusion, the okara under optimal fermentation operations could effectively reduce the pH value of okara and improved its preservation in both laboratory-scale and larger-scale fermentation, and it also benefited to maintain the nutritional value of okara. According to the in vitro and in situ results, replacing soybean meal with different ratios of fermented okara had no effect on the in vitro digestibility, rumen degradation rate, and effective degradability. It suggested that the fermented okara had the potential to replace soybean meal in dairy cow diets as a stable alternative protein source.